Conditionally immortalized long-term stem cells and methods of making and using such cells

ABSTRACT

Disclosed are methods for conditionally immortalizing stem cells, including adult and embryonic stem cells, the cells produced by such methods, therapeutic and laboratory or research methods of using such cells, and methods to identify compounds related to cell differentiation and development or to treat diseases, using such cells. A mouse model of acute myeloid leukemia (AML) and cells and methods related to such mouse model are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/583,970, entitled “CONDITIONALLY IMMORTALIZED LONG-TERM STEM CELLSAND METHODS OF MAKING AND USING SUCH CELLS” filed Oct. 18, 2006, whichis herein incorporated by reference in its entirety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C.§119(e)from U.S. Provisional Patent Application No. 60/728,131, filed Oct. 18,2005, and from U.S. Provisional Application No. 60/765,993, filed Feb.6, 2006. The entire disclosure of each of U.S. Provisional PatentApplication No. 60/728,131 and U.S. Provisional Patent Application No.60/765,993 is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 10, 2010, is named36824701.txt, and is 121,120 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to conditionally immortalizedlong term stem cells, to methods of producing such cells, and to methodsof using such cells, including therapeutic methods and drug discoverymethods.

BACKGROUND OF THE INVENTION

The ability to manipulate the bone marrow output of various blood cellshas become an important tool in the management of several diseases. Someof the best new therapies for hematological malignancies are based onthe development of compounds that push leukemic cells to differentiateinto lineages to which they are committed prior to the transformingevent. One such example is the case of acute promyelocytic leukemia.Upon treatment of patients with Arsenic Trioxide, the malignant cellsare pushed along the myelomonocytic pathway leading to remission ofthose tumors. Another example lies in promotion of successfulengraftment of transplanted bone marrow stem cells (long termreconstituting hematopoietic stem cells, or lt-HSC) in irradiatedindividuals. The appearance of differentiated blood cells can beaccelerated by the systemic administration of cytokines that are knownto specifically induce red blood cell development (erythropoietin, orEpo), or myeloid cell development (granulocyte-macrophagecolony-stimulating factor, or GM-CSF). Finally, harvesting of lt-HSCfrom donors has been greatly simplified by the process of “mobilization”wherein these cells are induced to move from the bone marrow sites wherethey normally reside into peripheral blood by systemic administration ofa cytokine called G-CSF. Stem cells can then by harvested fromperipheral blood obviating the painful and elaborate collection of bonemarrow biopsies. All of these processes rely on the ability to programand control the biological behavior of lt-HSC.

Accordingly, bone marrow (stem cell) transplantation is an invaluabletherapeutic tool for hematologic and immune reconstitution ofindividuals who have undergone radiation and/or chemotherapy (e.g.cancer patients, or have been exposed to high-level radiation), and isalso a critical modality for treatment of immune deficiency andhematological malignancies. In addition, bone marrow transplantationwould be a highly useful therapy to combat the negative effects of agingon the immune system, as well as on other cells and tissues. It isestimated that stem cell transplantation could benefit more than 35,000children and adults per year.

The operative principle behind bone marrow transplantation isreplacement of radiation sensitive lt-HSC that give rise to all bloodcell types. Recent studies indicate that bone marrow transplantation mayhave value in the treatment of heart disease. Although the basis of thisaffect is unknown, it, and other findings, raise the possibility thathematopoietic stem cells (lt-HSC) may be reprogrammed to give rise toother tissues. If this is true, lt-HSC may have much broader utility andprovide an alternative to controversial embryonic stem cell therapy.

The major obstacles confronting clinical application of bone marrowtransplantation lie first in identification of an appropriatelyhistocompatible marrow donor. This is usually accomplished usingregistries that have enrolled more than 6 million potential donors. Theselected donor must undergo a grueling ordeal of induced mobilizationstem cell into the blood followed by 4-5 days of leukapheresis toisolate rare lt-HSC. Transplantation of these cells must be followed bycareful monitoring and treatment of the recipient to minimize graftversus host reactions caused by passenger lymphocytes.

Elucidation of the molecular basis of the impairment in hematopoieticlineage development has been complicated historically by the lowfrequency of relevant cell populations, which prevents biochemicalanalysis of signaling and downstream responses. In fact, this has been amajor limiting factor in all studies of hematopoiesis. In addition, thelimited availability of long-term hematopoietic stem cells (LT-HSCs) hasalso been a major obstacle in the treatment of many types of cancer aswell as several kinds of immune deficiencies in humans. To the best ofthe present inventors' knowledge, there are currently no available celllines that arose spontaneously that resemble lt-HSCs and candifferentiate into normal lineages in vitro, or that can reconstitutelethally irradiated mice or sub-lethally irradiated humans, nor have anymethods been described to deliberately generate such cell lines.Moreover, there are currently no viable technologies to continuouslyexpand lt-HSCs, such that these cells need to be obtained from a donorevery time they are needed.

There is also a dire need for additional modalities to treathematological malignancies and immune deficiency, and novel cytokines toincrease the output of transplanted lt-HSC. In addition, an appropriateplatform for target identification and drug discovery does not currentlyexist. The missing elements are cell lines that represent differentdevelopmental stages in hematopoietic lineages. Optimally, such cellsshould retain the ability to undergo further differentiation in aspecific lineage. Such cell lines are essential for identification ofgene products, and thus new drugable targets, involved in regulation ofcell development, proliferation and survival. In addition, such celllines are essential for the screening of small molecule and shRNAlibraries for loss-of function studies, as well as cDNA libraries forgain of function studies, in search of novel drugs.

Barriers to current drug discovery in this area include: (a) isolationof a sufficient number of cells from a particular developmental stage;(b) propagation of the cells in vitro for a sufficient length of time;and (c) ability to use conditional oncogenes to screen for drugs thatcould affect leukemic cells, and not normal HSCs or progenitors.

Therefore, there is a great need in the art for a method to generatelt-HSC cell lines that can be expanded extensively, frozen, and usedagain whenever they are required, in the absence of subsequent harvestsfrom the donor.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method to produceconditionally immortalized adult stem cells. The method includes thesteps of: (a) obtaining an expanded population of adult stem cells; (b)transfecting the stem cells with a nucleic acid molecule comprising aprotooncogene or biologically active fragment or homologue thereof thatpromotes cell survival and proliferation, wherein the protooncogene isinducible; (c) transfecting the stem cells with a nucleic acid moleculeencoding a protein that inhibits apoptosis of the cell; and (d)expanding the transfected cells in the presence of a combination of stemcell growth factors under conditions whereby the protooncogene isactive, to produce conditionally immortalized adult stem cells. In oneaspect of this embodiment, the nucleic acid molecule of (b) and/or (c)is contained in an integrating vector. In one aspect, the nucleic acidmolecule of (b) and/or (c) is transfected into the cells using a virusor viral vector selected from: retroviral vectors, lentivirus vectors,parvovirus, vaccinia virus, coronavirus, calicivirus, papilloma virus,flavivirus, orthomixovirus, togavirus, picornavirus, adenoviral vectors,modified and attenuated herpesviruses. In one aspect, the nucleic acidmolecule of (b) and/or (c) is transfected into the cells using directelectroporation. In one aspect, the nucleic acid molecule or (b) and/or(c) is contained in a vector comprising a nucleic acid sequence encodinga drug-sensitivity protein. In one aspect, the nucleic acid molecule or(b) and/or (c) is contained in a vector comprising nucleic acidsequences encoding recognition substrate sequences for a recombinaseflanking the nucleic acid molecule of (b) or (c).

In one aspect, this embodiment includes the additional steps of: (e)removing the conditions of (d) whereby the protooncogene is active; and(f) culturing the cells of (e) in media comprising growth factors thatinduce differentiation of the cells. This method can further include:(g) adding to the cells of (f), the conditions of (d) whereby theprotooncogene is active, to produce conditionally immortalized cells inan intermediate stage of cell differentiation.

Another embodiment of the present invention relates to a method toproduce conditionally immortalized adult stem cells, comprising: (a)obtaining an expanded population of adult stem cells; (b) culturing thestem cells in the presence of: (1) a combination of stem cell growthfactors; (2) a first Tat-fusion protein, wherein Tat is fused to aprotein encoded by a protooncogene or biologically active fragment orhomologue thereof that promotes cell survival and proliferation; and (3)a second Tat-fusion protein, wherein Tat is fused to a protein thatinhibits apoptosis in the stem cells.

Yet another embodiment of the present invention relates to method toproduce conditionally immortalized embryonic stem cells, comprising: (a)obtaining an expanded population of embryonic stem cells; (b)transfecting the stem cells with a nucleic acid molecule comprising aprotooncogene or biologically active fragment or homologue thereof thatpromotes cell survival and proliferation, wherein the protooncogene isinducible; (c) transfecting the stem cells with a nucleic acid moleculeencoding a protein that inhibits apoptosis of the cell; and (d)expanding the transfected cells in the presence of a combination of stemcell growth factors under conditions whereby the protooncogene isactive, to produce conditionally immortalized embryonic stem cells.

Another embodiment of the present invention relates to method to produceconditionally immortalized stem cells, comprising: (a) obtaining anexpanded population of stem cells; (b) culturing the stem cells in thepresence of: (1) a combination of stem cell growth factors; (2) aprotein encoded by a protooncogene or biologically active fragment orhomologue thereof that promotes cell survival and proliferation; and;(3) a protein that inhibits apoptosis in the stem cells. The protein of(2) and (3) are delivered into the stem cells using any suitabledelivery system, including, but not limited to, Tat fusion, aptamerstechnology, or CHARIOT™ technology.

Yet another embodiment of the present invention relates to a method toproduce conditionally immortalized stem cells, comprising: (a) obtainingan expanded population of stem cells; (b) delivering into the cells aprotein encoded by a protooncogene or biologically active fragment orhomologue thereof that promotes cell survival and proliferation, or anucleic acid molecule encoding the same, wherein the protooncogene isinducible; (c) inhibiting apoptosis in the stem cells by delivering intothe cells a protein that inhibits apoptosis of the cell, a nucleic acidmolecule encoding the protein that inhibits apoptosis of the cell, or anucleic acid molecule or protein that inhibits a proapoptotic protein inthe cells; and (d) expanding the cells in the presence of a combinationof stem cell growth factors under conditions whereby the protooncogeneis active, to produce conditionally immortalized adult stem cells.

In any of the embodiments described above, the protooncogene can beselected from, but is not limited to: MYC-ER and ICN-1-ER. In any of theembodiments described above, the protein that inhibits apoptosis can beselected from, but is not limited to a member of the Bcl-2 family thatinhibits apoptosis, such as Bcl-2, Bcl-X, Bcl-w, BclXL, MeI-1, Dad-1, orhTERT. When the protooncogene is MYC-ER or ICN-1-ER, the conditionsunder which the protooncogene is active can include the presence oftamoxifen or an agonist thereof. In one aspect the cells are transfectedwith or are delivered (as a protein) MYC-ER and Bcl-2; MYC-ER and hTERT;ICN-1-ER and Bcl-2; ICN-1-ER and hTERT; or MYC-ER and ICN-1-ER.

In any of the embodiments described above, the step of expanding can beconducted in a medium including, but not limited to, (1) interleukin-6(IL-6), IL-3 and stem cell factor (SCF); (2) a serum-free mediumcomprising stem cell factor (SCF), thrombopoietin (TPO), insulin-likeGrowth Factor 2 (IGF-2) and fibroblast Growth Factor 1 (FGF-1).

In any of the embodiments described above, the adult stem cells caninclude, but are not limited to: hematopoietic stem cells, intestinalstem cells, osteoblastic stem cells, mesenchymal stem cells, neural stemcells, epithelial stem cells, cardiac myocyte progenitor stem cells,skin stem cells, skeletal muscle stem cells, and liver stem cells. Inone aspect, the mesenchymal stem cells are selected from lungmesenchymal stem cells and bone marrow stromal cells. In one aspect, theepithelial stem cells are selected from the group consisting of lungepithelial stem cells, breast epithelial stem cells, vascular epithelialstem cells and intestinal epithelial stem cells. In one aspect, the skinstem cells are selected from the group consisting of epidermal stemcells and follicular stem cells (hair follicle stem cells). In oneaspect, the neural cells are selected from neuronal dopaminergic stemcells and motor-neuronal stem cells. In one aspect, the stem cells arefrom fresh or cryopreserved cord blood. In one aspect, the stem cellsare hematopoietic progenitor cells obtained from the peripheral blood ofnormal or granulocyte colony-stimulating factor (G-CSF) treatedpatients.

In any of the embodiments described above, the method can furtherinclude genetically modifying the stem cells to correct a genetic defectin the cells, genetically modifying the stem cells to silence theexpression of a gene, and/or genetically modifying the stem cells tooverexpress a gene.

In any of the embodiments described above, the method can furtherinclude storing the cells. In one aspect, the method further includesretrieving the cells from storage and culturing the cells.

Another embodiment of the present invention relates to cells produced byany method described above or elsewhere herein.

Yet another embodiment of the present invention relates to a method toprovide adult stem cells, or cells differentiated therefrom, to anindividual comprising: (a) providing a source of conditionallyimmortalized adult stem cells produced by any method described above orelsewhere herein; (b) removing the conditions under which the stem cellsof (a) are conditionally immortalized; and (c) administering the stemcells or cells differentiated therefrom to the individual. In oneaspect, the cells were previously obtained from the individual in (c).In one aspect, the cells were obtained from a previously frozen stock ofsaid cells. In one aspect, the cells are freshly obtained from theindividual and conditionally immortalized by any method described aboveor elsewhere herein. In one aspect, the individual has cancer. Inanother aspect, the individual has leukemia. In another aspect, theindividual has an immune deficiency disorder. In another aspect, theindividual has an anemia disorder. In another aspect, the individual isundergoing reconstructive surgery. In another aspect, the individual isundergoing elective cosmetic surgery. In another aspect, the individualis undergoing transplantation surgery. In one aspect, the individual isin need of stem cells, or cells differentiated therefrom, selected from:hematopoietic stem cells, intestinal stem cells, osteoblastic stemcells, mesenchymal stem cells, neural stem cells, epithelial stem cells,cardiac myocyte progenitor stem cells, skin stem cells, skeletal musclestem cells, and liver stem cells. In another aspect, the individual isin need of improved immune cell function. In another aspect, theindividual has a genetic defect that is corrected by the stem cell.

Yet another embodiment of the present invention relates to a method toidentify compounds that regulate lineage commitment and/or celldifferentiation and development, comprising: (a) contacting adult stemcells produced by any method described above or elsewhere herein; and(b) detecting at least one genotypic or phenotypic characteristic in thestem cells of (a), as compared to the stem cells in the absence of thecompound, wherein detection of a difference in the characteristic in thepresence of the compound indicates that the compound affects thecharacteristic in the stem cell.

Another embodiment of the present invention relates to a method to studylineage commitment and/or cell differentiation and development,comprising evaluating adult stem cells produced by any method describedabove or elsewhere herein, or cells differentiated therefrom, to detectat least one genotypic or phenotypic characteristic of the cells.

Yet another embodiment of the present invention relates to the use ofthe cells produced by any method described above or elsewhere herein ina medicament for treating a condition or disease in whichtransplantation of stem cells is beneficial.

Another embodiment of the present invention relates to a mouse model ofacute myeloid leukemia (AML), comprising a mouse produced by a methodcomprising: (a) lethally irradiating a mouse; (b) transferringconditionally immortalized long-term stem cells produced by any methoddescribed above or elsewhere herein and whole bone marrow cells from aRag^(−/−) mouse into the mouse; and (c) injecting periodic doses oftamoxifen or an agonist thereof into the mouse until the mouse developsclinical signs of AML. In one aspect, the cells are transfected with orare delivered (as a protein) MYC-ER and Bcl-2.

Another embodiment of the invention relates to tumor cells obtained fromthe mouse model of AML described above.

Yet another embodiment of the invention relates to the use of the mousemodel of AML for preclinical testing of drug candidates specific forhuman proteins; to identify, develop, and/or test a compound for use inthe diagnosis of, study of, or treatment of AML; or to identify,develop, and/or test a target for use in the diagnosis of, study of, ortreatment of AML.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIG. 1 is a graph showing mortality curves following bone marrowtransplantation of transduced cells and activation of MYC function with4OHT, in vivo.

FIG. 2 is a scatter plot showing scatter characteristics and GFPexpression levels of HSCs derived from young and aged mice, following invitro transduction. The dot plots represent the flow cytometric data forthe forward (FSC) and side (SSC) scatter characteristics of the HSCsafter three days in culture with IL-3, IL-6 and SCF. These two criteriacorrelate with cell size (FSC) and granularity (SSC).

FIG. 3 is a scatter plot showing the phenotypic comparison of cell linesderived from irradiated recipients reconstituted using BCL-2, MYC-ER andEGFP-transduced hematopoietic stem cells from aged (>60% ID⁻ repertoire)and young 3-83μδ transgenic mice. Shown is the phenotype ofrepresentative clones 3 (young) and 3 (aged) months after initiation ofculture.

FIG. 4 is a scatter plot showing the spontaneous differentiation of theaged LT-HSC line (ABM46) in vitro following withdrawal of tamoxifen(stem cell and B lineage marker expression are analyzed by flowcytometry).

FIG. 5 is a scatter plot showing the analysis of hematopoietic cellcompartments derived from LT-HSC lines 6 weeks after adoptive transferinto irradiated young recipients. Data from three mice are presented inthis figure, one mouse received the aged HSC line ABM42, and two micereceived aged HSC line ABM46.

FIG. 6 is a scatter plot showing that the development of the B-cellcompartment is compromised in mice reconstituted with ABM42 and ABM46cell lines. Data from three mice are presented in this figure, one mousereceived the aged HSC line ABM42, and two mice received aged HSC lineABM46.

FIG. 7 is a scatter plot showing T-cell development in mice that werereconstituted with ABM42 and ABM46 cell lines. Data from three mice arepresented in this figure, one mouse received the aged HSC line ABM42,and two mice received aged HSC line ABM46.

FIG. 8 is a scatter plot and graph showing the phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with BCL-2 and MYC-ER and maintained incontinuous in vitro culture for >90 days. The panels represent theresults of the flow cytometric analysis for expression of the viralexpression markers (GFP and Thy1.1), as well as four markers required todefine long-term HSCs in mice, Sca-1, c-kit, CD34 and Flk-2. The fourcell lines contained subpopulations that retained the phenotypes oflt-HSCs (Sca-1+, c-kit+, CD34−, flk-2−).

FIG. 9 is a scatter plot and graph showing a phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in, continuous in vitro culture for >90 days (pMIG-MYC andpMIT-Bcl-2 (top panels), pMIG-MYC.ER and pMIG-hTERT (middle panels), orpMIG-ICN.1.ER and pMIT-Bcl-2 (bottom panels)).

FIG. 10 is a scatter plot and graph showing a phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days (pMIG-ICN.1.ERand pMIT-Bcl-2 (top panels), pMIG-ICN.1 and pMIT-Bcl-2 (second rowpanels), or pMIG-ICN.1 and pMIG-Bcl-2 (third row panels), or pMIG-hTERTand pMIT-Bcl-2 (bottom panels)).

FIG. 11 is a scatter plot and graph showing a phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days (pMIG-MYC andpMIG-ICN.1 (top panels), pMIG-MYC.ER and pMIG-ICN.1 (middle panels), orpMIG-ICN.1.ER and pMIG-MYC (bottom panels)).

FIG. 12 is a scatter plot showing the in vivo reconstitution of T celland 13 cell compartments from cell lines derived from HSCs obtained fromyoung C57/BL6 mice that were retrovirally transduced with differentcombinations of oncogenes and maintained in continuous in vitro culturefor >90 days.

FIG. 13 is a schematic drawing showing the use of recognition substratesequences (RSS's) for recombinases in order to ensure the excision ofrecombinant DNA from conditionally immortalized long-term stem cells ofthe invention prior to transplantation.

FIG. 14 is a graph showing the detection of cells of the NK anderythroid lineage differentiated from conditionally immortalizedlong-term stem cells of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a solution to the problem of being ableto generate, maintain and manipulate stable cell lines derived fromlong-term stem cells, and particularly, long-term hematopoietic stemcells (lt-HSCs), that can give rise to all cell lineages that wouldnormally arise from such cells when placed under the appropriateconditions. The present invention generally relates to methods toproduce conditionally immortalized, long-term stem cells, to the stemcells produced by such methods, and to methods of using such stem cells.More specifically, using long-term hematopoietic stem cells as anexemplary stem cell population, the present inventors have established apowerful method to produce stem cells that are conditionallyimmortalized (e.g., reversibly immortalized or immortalized underspecified conditions which is reversible when such conditions areremoved), such stem cells being capable differentiating into normal celllineages in vitro and in vivo, and being capable of reconstitutingsubjects in need of such cells. Indeed, the present invention caneliminate the need for a bone marrow donor, since the invention providesfor the ability to harvest stem cells from a patient prior to aprocedure (e.g., chemotherapy, radiation, etc.), to expand such cells,and return them to the patient. Moreover, such stem cells can beexpanded extensively, stored (e.g., frozen), and then retrieved andexpanded again, manipulated, and/or used repeatedly as required ordesired. Such stem cells can be manipulated, for example, to correct agenetic defect or provide a benefit to a subject (therapeutic orpreventative), or differentiated into a desired cell type. Finally, suchcells can be used in a variety of assays for the identification of newtargets involved in regulation of cell development, proliferation andsurvival, and the identification and development of drugs useful inameliorating or treating diseases and conditions that would benefit fromthe regulation of cell development, proliferation and/or survival.

The present inventors have developed novel technology that allows theconditional immortalization of long-term stem cells, exemplified hereinby long-term hematopoietic stem cells (lt-HSCs). The resulting celllines can be expanded (propagated) indefinitely and exponentially invitro and/or cryopreserved (stored), and have the ability to rescuelethally irradiated mice and to reconstitute all blood cell lineages insuch animals. Furthermore, the inventors have been able to generatedifferentiated blood cells in vitro by extinguishing the function of thetransforming oncogene. Such cells and the methods of producing them asdescribed herein will allow the generation of transplantable human stemcells that carry no recombinant DNA, and thus pose no long term risk tothe recipient. These conditionally immortalized lt-HSC's of theinvention can be stabilized in their mature phenotypes and cell linesestablished in which the mature phenotype is preserved afterreactivation of the oncogene. For example, the inventors have been ableto develop CD4⁺ αβ⁺ T cells, as well as dendritic cell lines.

Applied in the clinical setting, this technology has the followingadvantages over bone marrow transplantation:

1. Very few lt-HSC are needed to establish clones;

2. Clones represent a renewable resource that can be stored indefinitelyand accessed quickly;

3. The cost of this therapy should be much less than conventional bonemarrow transplantation;

4. Use of lt-HSC clones should mitigate the threat of graft-versus-hostdisease, and associated costs;

5. This technology can, at least in some cases, mitigate the need for abone marrow donor.

In addition, the present invention provides for the use of theconditionally transformed long-term stem cells, such as the lt-HSCcells, to generate cells representing differentiated lineages (e.g.,differentiated hematopoietic lineages, including intermediate stages ofdevelopment of hematopoietic lineages). For example, in addition tocountless therapeutic and preventative applications, these cell lineswill allow the identification of novel compounds that can inducedifferentiation of malignant cells, arrest their growth, or induceapoptosis. These cells will also permit screening for novel cytokinesand growth factors that direct the differentiation of stem cells in aparticular pathway. Such cell lines simply do not exist and will beessential for drug discovery.

More specifically, in an effort to overcome the limitations in the artwith regard to the provision and use of long term populations ofadult-derived stem cells (although the invention is not limited toadult-derived stem cells, as discussed below), the present inventorshave developed novel methods of producing of conditionally transformedcell lines representing early hematopoietic stem cell progenitors. In aspecific, non-limiting example of the technology described andexemplified herein, the strategy involved the transfection (e.g., byretroviral transduction) of bone marrow stem cells from 5-fluorouracil(5-FU)-treated 3-83μδ mice. The inventors utilized the pMSCV bisistronicretroviral vector with inserts encoding Bcl-2 and green fluorescentprotein (GFP) (as a reporter gene), and MYC-ER and GFP (again as areporter gene). MYC was selected because of its ability to substitutefor cytokine-derived survival and proliferative signals in lymphocytes.By restricting the target cell, the inventors hypothesized that stemcell tumors would form. Importantly, MYC-ER function is tamoxifendependent in this setting, allowing for the termination of MYC functionand transformation by withdrawing tamoxifen from the animal or cultures.In cells transduced with MYC-ER, the fusion protein is produced, but isretained in the cytoplasm until exposed to tamoxifen. Bcl-2 was selectedbecause of its ability to inhibit apoptosis of cells that would normallyoccur as a result of exposure to the MYC signals and more particularly,when MYC is “inactivated” or removed by withdrawal of the tamoxifen fromthe cells. This novel combination of gene types (i.e., the invention isnot limited to these specific genes, as discussed in more detail below)is partly responsible for the successful production of conditionallyimmortalized stem cells according to the present invention, and canreadily be extended to other similar combinations of genes, as discussedin detail below.

Recipients of the transduced stern cells described above produced tumors(in the presence of 4OHT), and tumor cells from the bone marrow, spleenand lymph node were harvested and placed in culture with tamoxifen and astem cell growth factor cocktail. The present inventors have discoveredthat, in the absence of an appropriate combination of stem cell growthfactors, the stem cells produced by the present method will stop growingand die within a short period of time. Therefore, the use of a stem cellgrowth factor “cocktail” (i.e., combination of appropriate or suitablegrowth factors for stem cells) after transfection of the cells with thecombination of genes discussed above is a second important aspect of themethod of the present invention. This cocktail, while having the generalcharacteristic of promoting and maintaining the growth of the stemcells, is not limited to a particular combination of growth factors, andparameters for selection of such factors are discussed in detail below.

The stem cells generated by the method of the present invention could beexpanded in culture and were homogeneously positive for, e.g., Sca1,positive for Endoglin and ckit, and negative for CD34, Flt3, B220, CD19and mIgM, which are indicative of the phenotype of lt-HSC, which iswell-characterized in the art. These cells could be frozen(cryopreserved, or stored), and then easily recovered and cultured afterfreezing. Importantly, the recovered cells were homogenous in phenotypeand exhibited the phenotype of lt-1-ISC (e.g., again, uniformly GFPbright cells were positive for Scat, Endoglin and ckit, and negative forCD34, Flt3, B220, CD19 and mIgM). This phenotype corresponds perfectlywith the published characteristics of long term repopulating pluripotentstem cells (Reya et al., 2003, Nature 423:409-14) that provide alllong-term reconstitution in mice.

The inventors have further developed this method so that it can beperformed completely in vitro (i.e., the initial procedure was conductedpartly in vivo as described above). The inventors have also demonstratedthat other combinations of genes having similar characteristics as thosedescribed above also result in the conditional immortalization oflt-HSCs. Furthermore, the cell lines can be differentiated in vitro intohematopoietic lineages by removing the tamoxifen and providing theappropriate growth factors, and will differentiate in vivo into allhematopoietic lineages in recipient animals in which tamoxifen iswithheld. In addition, the cells can be differentiated into intermediatelevels of development that have a stable phenotype and retain theirability to further differentiate along their committed pathway uponapplication or removal of the appropriate signal (described herein).Such cells are invaluable for various therapeutic applications. All ofthese experiments are described in detail below and in the Examples.

The methods and cell lines of the present invention provide a uniqueopportunity not only to study in detail the molecular, biochemical andcellular events that are associated with the commitment of adult stemcells toward various cell lineages and to study the differentiation anddevelopment of stem cells into various cell lineages, but also provideunique therapeutic and drug discovery tools.

For example, the stem cell lines of the present invention provide aunique source of expandable stem cells for use in a variety oftransplantation, therapeutic and preventative strategies, including thetreatment of cancer, and particularly, cancer that is treated byradiation. In current therapy for leukemia, for example, limited accessto bone marrow donors and finite supplies of stem cells from such donorsseverely limit the options for reconstitution of a patient afterradiation therapy. The present invention solves this problem byproviding a means to generate a continuously expandable and renewablesupply of autologous stem cells or histocompatible stem cells that canbe stored and recovered as needed. Such technology could ultimatelyablate the need for bone marrow donors altogether. In addition, avariety of immune deficiency disorders and anemia disorders (e.g.,aplastic anemia or hemolytic anemia) will also benefit greatly from thistechnology, since the present invention provides the ability torepopulate hematopoietic cells of an individual as needed by theindividual. Furthermore, the aging process is associated with severalimportant changes in the hematopoietic compartment, including theincreasing inability to mount a productive immune response, amongothers. Hematopoietic stem cells from aged mice have been shown tocontain a higher level of mRNAs for DNA-repair problems. This mayultimately affect their ability to self-renew, undergo differentiation,undergo proliferation, and survive in response to bone marrow cytokines.Therefore, an aging individual can also benefit from the presentinvention in that a continuous supply of healthy hematopoietic cells canbe provided to correct or ameliorate such deficiencies.

The technology of the present invention is not limited to bone marrowstem cells, but can be applied to virtually any type of stem cell, andcan be extended beyond adult-derived cells to embryonic stem cells.

In one example, another application of the present invention relates tothe generation of continuously expandable and renewable hair folliclestem cells. The development of conditionally immortalized stem cellsfrom this lineage can be use in the context of reconstructive surgeryfor burn victims, for any individual that undergoes chemotherapy and/orradiation therapy resulting in the irreversible loss of hair growth, aswell as patients following any surgical procedure affecting the skull.Furthermore, such cells could be used for elective procedures thatinvolve the induction of hair growth in individuals affected byhereditary pattern baldness. Similarly, application of the presentinvention to stem cells of the skin will be invaluable for use in woundhealing and treatment of burn victims, as well as plastic reconstructivesurgery for trauma and other patients, as well as elective surgeries,including, but not limited to, cosmetic surgery. Such cells can beadditionally genetically manipulated to correct inborn or acquiredgenetic defects in young and aged individuals. One of skill in the artwill understand based on this disclosure that benefits can be derivedfrom the use of the present invention on various other stem cellpopulations, including, but not limited to, stem cells derived fromlung, breast, and intestinal epithelium and stem cells derived fromneural and cardiac tissue, to name just a few. Other stem cell types arereferenced elsewhere herein.

In addition, the present invention provides the unique opportunity foran individual to have access to expandable supplies of autologous stemcells and cells differentiated therefrom as needed throughout the lifeof the individual. For example, as the body ages, it is known thatimmune function and immune memory deteriorates. However, using thetechnology provided by the present invention, it will be possible torepopulate an individual with new, autologous stem cells that arecapable of differentiation into all of the cells of the hematopoieticlineage, thus providing the aged individual with a “young” immunesystem. In addition, stem cells generated by the present method can bestored and used as part of therapeutic protocols during the lifetime ofthe individual, should they be needed (e.g., in the event the individualdevelops a cancer or immune deficiency disease or has another need fornewly generated, autologous cells of virtually any type).

The present invention also provides unique opportunities for genetherapy. Specifically, genetic defects can now be corrected orbeneficial gene modifications can be introduced into somatic cells bymanipulating autologous stem cells obtained from an individual that havebeen conditionally immortalized and expanded using the method of thepresent invention. The stem cells can then be reintroduced into theindividual from which they were obtained.

The stem cells produced by the method of the invention can also be usedin a variety of drug discovery assays. Since one can now producevirtually unlimited supplies of homogeneous stem cells that can readilybe stored, recovered, expanded and manipulated, such stem cells can beused as stem cells or differentiated into various cell lineages and usedin assays to test various compounds for effects on cell differentiation,gene expression, and cell processes. The cells can be manipulated priorto contact with the compounds, such as by genetic manipulation. Stemcells from individuals with genetic defects can be evaluated in suchassays in order to identify therapeutic compounds (e.g., cancertherapeutics) and evaluate gene replacement therapies. Indeed, thetechnology of the present invention provides an opportunity to targetthe cells of a specific individual to identify drug candidates andtherapeutic candidates and strategies that are “tailored” to the cellsof an individual. An example of such an assay is described in detailbelow.

With regard to research and discovery in the area of lineage commitmentand cell differentiation and development, prior to the presentinvention, such studies were severely hampered by the lack of access toand the inability to generate sufficient numbers of the desired cellpopulation to perform desired experiments. For example, in order toidentify or screen for intermediates in the differentiation of aparticular progenitor cell line, a sufficient number of cells must beobtained to provide meaningful and reproducible results. The progenitorcell line should also retain the ability to further differentiate in thelineage to which it has already committed, hence making these noveltools that do not currently exits, nor are there other descriptions oftechnology needed to generate those cells. Using technologies availableat the time of the invention, this was not possible. The presentinvention solves the problem by providing expandable and essentiallyunlimited supplies of homogeneous stem cells that can be used in avariety of experiments. This technology will greatly enhance researchcapabilities in the area of cell differentiation and discovery.

As discussed above, the method for conditionally immortalizing lt-HSCsof the present invention can be adapted for additional stem cellsderived from other tissues. For example, by adapting the gene deliveryand growth factors, if needed, the present invention can be applied to avariety of different stem cells as described below. Such cells can alsobe expanded in vitro, and proceed to differentiate upon inactivation ofthe oncogenes, as described herein for hematopoietic stem cells. Thesecells can then be used for therapeutic applications that include tissuerepair and tissue regeneration/engineering. Accordingly, the MYC-ER andBcl-2 combination of genes, or any of the other combinations describedherein, can be transfected by any method described herein or deemedsuitable by one of skill in the art given this disclosure (including bya variety of viral-mediated methods), into cells including, but notlimited to, mesenchymal stem cells (including, but not limited to, lungmesenchymal stem cells, bone marrow stromal cells), neural stem cells(including, but not limited to, neuronal dopaminergic stem cells andmotor-neuronal stem cells), epithelial stem cells (including, but notlimited to, lung epithelial stem cells, breast epithelial stem cells,and intestinal epithelial stem cells), cardiac myocyte progenitor stemcells, skin stem cells (including, but not limited to, epidermal stemcells and follicular stem cells), skeletal muscle stem cells,endothelial stern cells (e.g., lung endothelial stem cells), and liverstem cells, to generate conditionally immortalized cell lines that canbe expanded in vitro and proceed to differentiate upon inactivation ofthe oncogenes. In addition to the therapeutic potential of such celllines, these lines can be further modified in vitro (or ex vivo) inorder to correct inborn genetic defects, and used for studying themolecular basis of early lineage commitment and differentiation. Whilethese cells may be a novel source of potentially relevant therapeutictargets, these cell lines will also be useful for the screening of smallmolecules that either prevent or induce differentiation, and for theidentification of novel compounds and molecular targets for varioustherapies, including, but not limited to, cancer therapy and immunedeficiency therapy.

General Definitions

In accordance with the present invention, reference to an isolatednucleic acid molecule herein is a nucleic acid molecule that has beenremoved from its natural milieu (i.e., that has been subject to humanmanipulation), its natural milieu being the genome or chromosome inwhich the nucleic acid molecule is found in nature. As such, “isolated”does not necessarily reflect the extent to which the nucleic acidmolecule has been purified, but indicates that the molecule does notinclude an entire genome or an entire chromosome in which the nucleicacid molecule is found in nature. An isolated nucleic acid molecule caninclude a gene. An isolated nucleic acid molecule that includes a geneis not a fragment of a chromosome that includes such gene, but ratherincludes the coding region and regulatory regions associated with thegene, but no additional genes that are naturally found on the samechromosome. An isolated nucleic acid molecule can also include aspecified nucleic acid sequence flanked by (i.e., at the 5′ and/or the3′ end of the sequence) additional nucleic acids that do not normallyflank the specified nucleic acid sequence in nature (i.e., heterologoussequences). Isolated nucleic acid molecule can include DNA, RNA (e.g.,mRNA), or derivatives of either DNA or RNA (e.g., cDNA, siRNA, shRNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein or domain of a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect (e.g., provision ofan inducible protooncogene, as described herein).

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress (1989)). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule or polynucleotide of thepresent invention is a size sufficient to encode a protein useful in thepresent invention, such as a protein encoded by a protooncogene orfunctional portion thereof (i.e., a portion that has the biologicalactivity of the full-length protein and that is sufficient for use inthe method of the invention), or an anti-apoptotic protein or afunctional portion thereof (i.e., a portion that has the biologicalactivity of the full-length protein and that is sufficient for use inthe method of the invention). Other nucleic acid molecules that may beuseful in the present invention can include nucleic acid molecules of aminimum size sufficient to form a probe or oligonucleotide primer thatis capable of forming a stable hybrid with the complementary sequence ofa nucleic acid molecule encoding the natural protein (e.g., undermoderate, high or very high stringency conditions), which is typicallyat least 5 nucleotides in length, and preferably ranges from about 5 toabout 50 or about 500 nucleotides or greater, including any length inbetween, in whole number increments (i.e., 5, 6, 7, 8, 9, 10, . . . 33,34, . . . 256, 257, . . . 500). There is no limit, other than apractical limit, on the maximal size of a nucleic acid molecule of thepresent invention, in that the nucleic acid molecule can include asequence or sequences sufficient to be useful in any of the embodimentsof the invention described herein.

As used herein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C.′ below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

In one embodiment of the present invention, any amino acid sequencedescribed herein, including truncated forms (fragments or portions) andhomologues of such sequences, can be produced with from at least one,and up to about 20, additional heterologous amino acids flanking each ofthe C- and/or N-terminal end of the given amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” a given amino acid sequence. According to the presentinvention, the heterologous amino acids are a sequence of amino acidsthat are not naturally found (i.e., not found in nature, in vivo)flanking the given amino acid sequence or which would not be encoded bythe nucleotides that flank the naturally occurring nucleic acid sequenceencoding the given amino acid sequence as it occurs in the gene, if suchnucleotides in the naturally occurring sequence were translated usingstandard codon usage for the organism from which the given amino acidsequence is derived. Similarly, the phrase “consisting essentially of”,when used with reference to a nucleic acid sequence herein, refers to anucleic acid sequence encoding a given amino acid sequence that can beflanked by from at least one, and up to as many as about 60, additionalheterologous nucleotides at each of the 5′ and/or the 3′ end of thenucleic acid sequence encoding the given amino acid sequence. Theheterologous nucleotides are not naturally found (i.e., not found innature, in vivo) flanking the nucleic acid sequence encoding the givenamino acid sequence as it occurs in the natural gene.

According to the present invention, a recombinant vector (also referredto generally as a recombinant nucleic acid molecule, particularly whenit contains a nucleic acid sequence of interest according to theinvention) is an engineered (i.e., artificially produced) nucleic acidmolecule that is used as a tool for manipulating a nucleic acid sequenceof choice and for introducing such a nucleic acid sequence into a hostcell. The recombinant vector is therefore suitable for use in cloning,sequencing, and/or otherwise manipulating the nucleic acid sequence ofchoice, such as by expressing and/or delivering the nucleic acidsequence of choice into a host cell. Such a vector typically containsheterologous nucleic acid sequences, i.e., nucleic acid sequences thatare not naturally or usually found adjacent to a nucleic acid sequenceto be cloned or delivered, although the vector can also containregulatory nucleic acid sequences (e.g., promoters, untranslatedregions) which are naturally found adjacent to nucleic acid molecules ofthe present invention, or which are useful for expression of the nucleicacid molecules of the present invention (discussed in detail below). Avector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a plasmid or a viral vector. The vector can be maintainedas an extrachromosomal element (e.g., a plasmid) or it can be integratedinto the chromosome of a host cell. The entire vector can remain inplace within a host cell, or under certain conditions, the plasmid DNAcan be deleted, leaving behind the nucleic acid molecule of the presentinvention. Under other conditions, the vector is designed to be excised(removed) from the genome of the host cell at a selected time (describedin more detail below). The integrated nucleic acid molecule can be underchromosomal promoter control, under native or plasmid promoter control,or under a combination of several promoter controls. Single or multiplecopies of the nucleic acid molecule can be integrated into thechromosome. A recombinant vector of the present invention can contain atleast one selectable marker.

According to the present invention, the phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence (e.g., a transcription control sequence and/or a translationcontrol sequence) in a manner such that the molecule can be expressedwhen transfected (i.e., transformed, transduced, transfected, conjugatedor conduced) into a host cell. Transcription control sequences aresequences that control the initiation, elongation, or termination oftranscription. Particularly important transcription control sequencesare those that control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in a host cell or organism into which the recombinant nucleicacid molecule is to be introduced.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transduction” is a specific type of transfection in which geneticmaterial is transferred from one source to another, such as by a virus(e.g., a retrovirus) or a transducing bacteriophage. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as bacteria and yeast.In microbial systems, the term “transformation” is used to describe aninherited change due to the acquisition of exogenous nucleic acids bythe microorganism and is essentially synonymous with the term“transfection.” However, in animal cells, transformation has acquired asecond meaning that can refer to changes in the growth properties ofcells in culture after they become cancerous, for example. Therefore, toavoid confusion, the term “transfection” is preferably used herein withregard to the introduction of exogenous nucleic acids into animal cells.Therefore, the term “transfection” will be used herein to generallyencompass transfection or transduction of animal cells, andtransformation or transduction of microbial cells, to the extent thatthe terms pertain to the introduction of exogenous nucleic acids into acell. Transfection techniques include, but are not limited to,transformation, transduction, particle bombardment, diffusion, activetransport, bath sonication, electroporation, microinjection,lipofection, adsorption, infection and protoplast fusion.

As used herein, reference to an isolated protein or polypeptide in thepresent invention includes full-length proteins, fusion proteins,chimeric proteins, or any fragment (truncated form, portion) orhomologue of such a protein. More specifically, an isolated proteinaccording to the present invention, is a protein (including apolypeptide or peptide) that has been removed from its natural milieu(i.e., that has been subject to human manipulation), and can include,but is not limited to, purified proteins, partially purified proteins,recombinantly produced proteins, membrane bound proteins, proteinscomplexed with lipids, soluble proteins, synthetically producedproteins, and isolated proteins associated with other proteins. As such,“isolated” does not reflect the extent to which the protein has beenpurified. Preferably, an isolated protein of the present invention isproduced recombinantly. In addition, and again by way of example withrespect to the naming of a particular protein (Bcl-2), a “human Bcl-2protein” or a protein “derived from” a human Bcl-2 protein refers to aBcl-2 protein (including a homologue or portion of a naturally occurringBcl-2 protein) from a human (Homo sapiens) or to a Bcl-2 protein thathas been otherwise produced from the knowledge of the structure (e.g.,sequence) and perhaps the function of a naturally occurring Bcl-2protein from Homo sapiens. In other words, a human Bcl-2 proteinincludes any Bcl-2 protein that has substantially similar structure andfunction of a naturally occurring Bcl-2 protein from Homo sapiens orthat is a biologically active (i.e., has biological activity) homologueof a naturally occurring Bcl-2 protein from Homo sapiens as described indetail herein. As such, a human Bcl-2 protein can include purified,partially purified, recombinant, mutated/modified and syntheticproteins. According to the present invention, the terms “modification”and “mutation” can be used interchangeably, particularly with regard tothe modifications/mutations to the amino acid sequence of a protein (ornucleic acid sequences) described herein.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by modifications,including minor modifications, to the naturally occurring protein orpeptide, but which maintains the basic protein and side chain structureof the naturally occurring form: Such changes include, but are notlimited to: changes in one or a few (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or10) amino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a protein or truncated form of the protein or peptide),insertions and/or substitutions; changes in stereochemistry of one or afew atoms; and/or minor derivatizations, including but not limited to:methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have either enhanced,decreased, or substantially similar properties as compared to thenaturally occurring protein or peptide. A homologue can include anagonist of a protein or an antagonist of a protein.

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

In one embodiment, a homologue of a given protein comprises, consistsessentially of, or consists of, an amino acid sequence that is at leastabout 45%, or at least about 50%, or at least about 55%, or at leastabout 60%, or at least about 65%, or at least about 70%, or at leastabout 75%, or at least about 80%, or at least about 85%, or at leastabout 90%, or at least about 95% identical, or at least about 95%identical, or at least about 96% identical, or at least about 97%identical, or at least about 98% identical, or at least about 99%identical (or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein. In oneembodiment, the homologue comprises, consists essentially of, orconsists of, an amino acid sequence that is less than 100% identical,less than about 99% identical, less than about 98% identical, less thanabout 97% identical, less than about 96% identical, less than about 95%identical, and so on, in increments of 1%, to less than about 70%identical to the naturally occurring amino acid sequence of thereference protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

According to the present invention, an isolated protein, including abiologically active homologue or fragment thereof, has at least onecharacteristic of biological activity of activity the wild-type, ornatural (native) protein. In general, the biological activity orbiological action of a protein refers to any function(s) exhibited orperformed by the protein that is ascribed to the naturally occurringform of the protein as measured or observed in vivo (i.e., in thenatural physiological environment of the protein) or in vitro (i.e.,under laboratory conditions). Modifications, activities or interactionswhich result in a decrease in protein expression or a decrease in theactivity of the protein, can be referred to as inactivation (complete orpartial), down-regulation, reduced action, or decreased action oractivity of a protein. Similarly, modifications, activities orinteractions that result in an increase in protein expression or anincrease in the activity of the protein, can be referred to asamplification, overproduction, activation, enhancement, up-regulation orincreased action of a protein.

Method of Conditional Immortalization of the Invention

One embodiment of the present invention relates to a method to produceconditionally immortalized, adult stem cells, and preferably long-termstem cells. The method generally includes the following steps: (a)obtaining an expanded population of adult stem cells; (b) transfecting(transducing) the stem cells with a vector comprising a protooncogenethat promotes cell survival and proliferation, wherein the protooncogeneis regulatable (inducible, controllable), (c) transfecting (transducing)the stem cells with a vector encoding a protein that inhibits apoptosisof the cell; and (d) expanding the transfected cells in the presence ofa combination of stem cell growth factors under conditions whereby theprotooncogene is active. In one embodiment, the vector is an integratingvector. Cells produced by this method can be cultured, expanded, stored,recovered, used in therapeutic methods, used in research and discoverymethods, genetically manipulated, induced to differentiate by removingthe conditions whereby the protooncogene is active, and/or used in anyother method described herein or apparent to one of skill in the artgiven this disclosure. Steps (b) and (c) can be performed in any order.

According to the present invention, the phrase “conditionallyimmortalized” refers to cells that are immortalized (e.g., capable ofindefinite growth without differentiation in a cytokine dependentfashion, while maintaining their ability and potential to differentiateinto a number of different lineages under the appropriate conditions) ina reversible manner, such that the cells are immortalized under aspecific set of conditions, and when the conditions are removed orchanged (or other conditions added), the cells are no longerimmortalized and may differentiate into other cell types. The phrase“conditionally immortalized” can be used interchangeably with the phrase“reversibly immortalized”. For example, referring to the method of thepresent invention, the presence of the regulatable protooncogene thatpromotes cell survival and proliferation causes the cells to retain animmortalized phenotype when the stem cell is placed under conditionsthat allow the protooncogene to be activated (e.g., tamoxifen or anagonist thereof in the case of MYC-ER). In other words, the cells growand expand indefinitely in culture, and are maintained in anundifferentiated state under these specific conditions. When theseconditions are removed (e.g., the tamoxifen is removed with respect toMYC-ER), the stem cells are no longer immortalized and can differentiateinto various cell lineages given the appropriate environment (e.g., theappropriate combination of growth factors).

Reference to “stem cells”, as used herein, refers to the term as it isgenerally understood in the art. For example, stem cells, regardless oftheir source, are cells that are capable of dividing and renewingthemselves for long periods, are unspecialized (undifferentiated), andcan give rise to (differentiate into) specialized cell types (i.e., theyare progenitor or precursor cells for a variety of different,specialized cell types). “Long-term”, when used in connection with stemcells, refers to the ability of stem cells to renew themselves bydividing into the same non-specialized cell type over long periods(e.g., many months, such as at least 3 months, to years) depending onthe specific type of stem cell. As discussed herein, phenotypiccharacteristics of various long-term stem cells from different animalspecies, such as long-term hematopoietic stem cells (lt-HSC) are knownin the art. For example, murine lt-HSC can be identified by the presenceof the following cell surface marker phenotype: c-kit+, Sca-1+, CD34−,flk2− (see Examples). Adult stem cells include stem cells that can beobtained from any non-embryonic tissue or source, and typically generatethe cell types of the tissue in which they reside. The term “adult stemcell” may be used interchangeably with the term “somatic stem cell”.Embryonic stem cells are stem cells obtained from any embryonic tissueor source.

In one embodiment of the invention, the stem cells used in the presentinvention can include any adult stem cells obtained from any source. Inanother embodiment of the invention, stem cells can include embryonicstem cells. Stem cells useful in the present invention include, but arenot limited to, hematopoietic stem cells, mesenchymal stem cells(including, but not limited to, lung mesenchymal stem cells, bone marrowstromal cells), neural stem cells, epithelial stem cells (including, butnot limited to, lung epithelial stem cells, breast epithelial stemcells, vascular epithelial stem cells, and intestinal epithelial stemcells), intestinal stem cells, cardiac myocyte progenitor stem cells,skin stem cells (including, but not limited to, epidermal stem cells andfollicular stem cells (hair follicle stem cells)), skeletal muscle stemcells, osteoblastic precursor stem cells, and liver stem cells.

Hematopoietic stem cells give rise to all of the types of blood cells,including but not limited to, red blood cells (erythrocytes), Blymphocytes, T lymphocytes, natural killer cells, neutrophils,basophils, eosinophils, monocytes, macrophages, and platelets.

Mesenchymal stem cells (including bone marrow stromal cells) give riseto a variety of cell types, including, but not limited to bone cells(osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes),lung cells, and other kinds of connective tissue cells such as those intendons.

Neural stem cells in the brain give rise to its three major cell types:nerve cells (neurons) and two categories of non-neuronal cells,astrocytes and oligodendrocytes.

Epithelial stem cells in the lining of various tissues give rise toseveral cell types that form the epithelium in tissues.

Skin stem cells occur in the basal layer of the epidermis and at thebase of hair follicles. The epidermal stem cells give rise tokeratinocytes, which migrate to the surface of the skin and form aprotective layer, and the follicular stem cells can give rise to boththe hair follicle and to the epidermis. Other sources of adult stemcells will be known to those of skill in the art.

Embryonic stem cells can give rise to all tissues and cells of the body.

Methods for obtaining such stem cells and providing initial cultureconditions, such as a liquid culture or semi-solid culture medium, areknown in the art. The cells are initially expanded in vivo or in vitro,by contacting the source of the stem cells with a suitable reagent thatexpands or enriches such cells in the tissue source or in culture. Forexample, in the case of hematopoietic stem cells, the donor individualcan be treated with an agent that enriches for hematopoietic stem cellsand encourages such cells to proliferate without differentiation, suchas 5-fluorouracil. Other suitable agents for expansion of a desired stemcell type will be known to those of skill in the art. Alternatively, andpreferably, adult stem cells are isolated from a tissue source and thenexpanded or enriched in vitro by exposure to a suitable agent. Forexample, with regard to hematopoietic stem cells, a method for producingan expanded culture of adult hematopoietic progenitors is described inVan Parijs et al., (1999; Immunity, 11, 763-70). Cells are obtained froman individual by any suitable method for obtaining a cell sample from ananimal, including, but not limited, to, collection of bone marrowcollection of a bodily fluid (e.g., blood), collection of umbilical cordblood, tissue punch, and tissue dissection, including particularly, butnot limited to, any biopsies of skin, intestine, cornea, spinal cord,brain tissue, scalp, stomach, breast, lung (e.g., including lavage andbronchioschopy), fine needle aspirates of the bone marrow, amnioticfluid, placenta and yolk sac.

In one embodiment, cells useful in the invention can also be obtainedfrom fresh, or cryopreserved (stored) cord blood, hematopoieticprogenitor populations that can be derived from the directeddifferentiation of embryonic stem (ES) cells in vitro, hematopoieticstem cells (HSCs) obtained from the peripheral blood of normal orgranulocyte colony-stimulating factor (G-CSF)-treated patients who havebeen induced to mobilize their lt-HSCs to the peripheral circulation.

Once an expanded population of stem cells is obtained (made available,provided, or produced), the cells are transfected, either sequentially(in any order) or simultaneously, with: (1) a vector comprising aprotooncogene that promotes cell survival and proliferation, wherein theprotooncogene is regulatable (inducible, controllable), and (2) a vectorencoding a protein that inhibits apoptosis of the cell. Preferably, thevector is an integrating vector, defined herein as any vector that hasthe ability to integrate into the genome of a cell (e.g., a retroviralvector). Various vectors and methods of transfection are described indetail below. The protooncogene is regulatable (inducible orcontrollable), so that the protooncogene can be activated anddeactivated (i.e., turned on or turned off) as desired to eithermaintain the stem cell in an immortalized state or to allow it todifferentiate into a desired cell type. Protooncongenes can be selected,or designed, to be regulated by any suitable method, including inresponse to any condition, such as the presence or absence of a compoundor agent, temperature, or any other suitable condition. By way ofexample, the protooncogenes MYC-ER (the estrogen receptor (ER)-regulatedMYC) and ICN-1-ER (the ER-regulated intracellular portion of Notch-1)described herein are both inducible in the presence of tamoxifen. It isnoted that such genes can also be engineered to be responsive to otherdimerizing drugs, such as FK1012, altered forms of Rapamycin, or couldbe expressed from vectors that contain a tetracycline responsiveelement. The latter scenario regulates expression of the protein, notthe function of a polypeptide present in the cell. Other similarmodifications of this platform technology will be apparent to those ofskill in the art.

The protooncogene useful in the method of the present invention is anyprotooncogene that promotes cell survival and proliferation. Preferredprotooncogenes to use in the method of the invention include, but arenot limited to MYC, ICN-1, hTERT (reverse transcriptase component of thehuman telomerase), NMYC, S-MYC, L-MYC, Akt, (myrystylated). In addition,other suitable genes to use or methods of the invention or ways tomodify genes to achieve the desired result include, but are not limitedto use of downstream signaling effectors such as pyruvate dehydrogenasekinase 1 (PDK-1); mammalian target of Rapamycin (mTOR); loss ofphosphatase and tensin homologue (PTEN) by shRNA; Bcl-3, Cyclin D1,Cyclin D3, Bcl-10, Bcl-6, BCR-ABL (breakpoint cluster region fusion withABL) and its various mutant forms, constitutively active forms of Stat5and Stat3, AML1-ETO (fusion of acute myelogenous leukemia 1 andrunt-related transcription factor 1), MLL-ENL (mixed lineage leukemiaand eleven nineteen leukemia), Hox genes, activated forms of theinterleukin-3 (IL-3) receptor β chain, and other cytokine receptorchains (epidermal growth factor receptor (EGFR), c-kit, platelet-derivedgrowth factor receptor (PDGFR), etc.), as well as wnt (all mammalianforms), β-catenin, sonic hedgehog (shh-1 and all mammalian forms), bmi-1and c-jun (all mammalian forms). Also, the present invention includesinducing the loss (or inhibition) of cyclin kinase inhibitors by shRNA,including, but not limited to, p16, p19, p21 and p27. In one embodiment,the present invention includes the use of regulatable homologues of anyor such protooncogenes (e.g., MYC-ER or ICN-1-ER) or other genes. TheExamples describe the use of both MYC-ER or ICN-1-ER to successfullyproduce conditionally immortalized lt-HSC using the method of presentinvention.

The nucleic acid sequence encoding human MYC is represented herein asSEQ ID NO:1, which encodes an amino acid sequence represented herein asSEQ ID NO:2. The nucleic acid sequence encoding hTERT is representedherein as SEQ ID NO:3, which encodes an amino acid sequence representedherein as SEQ ID NO:4. The nucleic acid sequence encoding human ICN-1 isrepresented herein as SEQ ID NO:11, which encodes an amino acid sequencerepresented herein as SEQ ID NO:12. ICN-1 a portion of Notch-1, andspecifically, amino acids 1757-2555 from Notch-1 (see Aster et al., MolCell Biol. 2000 Oct.; 20(20):7505-15, incorporated herein by referencein its entirety). The nucleotide and amino acid sequence for MYC-ER areknown in the art and the MYC-ER protein is described in Soloman et al.,Oncogene. 1995 Nov. 2; 11(9):1893-7, incorporated herein by reference inits entirety. ICN-1-ER was created by the present inventors and thenucleic acid sequence encoding this protein is represented herein as SEQID NO:13, which encodes an amino acid sequence represented by SEQ IDNO:14.

Similarly, a preferred anti-apoptosis gene is Bcl-2, although othergenes that encode proteins that inhibit apoptosis and particularly,maintain cell survival when the protooncogene is inactivated in the stemcell, are included in the present invention. The nucleic acid sequenceencoding Bcl-2 alpha is represented herein as SEQ ID NO:5, which encodesan amino acid sequence of SEQ ID NO:6. Bcl-2 beta is represented hereinas SEQ ID NO:7, which encodes an amino acid sequence of SEQ ID NO:8. An“anti-apoptosis” gene is defined herein as any gene that encodes aprotein that can inhibit (reduce, prevent, decrease) a processassociated with apoptosis in a cell or promote (enhance, increase,stimulate, allow) cell survival, even in the presence of conditions thatcould induce apoptosis. Proteins associated with apoptosis, and thegenes encoding such proteins, are well-known in the art. Such othergenes include, but are not limited to, any genes in the Bcl-2 familythat will likely be important in the setting of conditionaltransformation of adult stem cells (i.e., not just hematopoietic stemcells). These genes include, but are not limited to, other pro-survivalmembers of the Bcl-2 family, such as Bcl-X, Bcl-w, BclXL, Mcl-1, Dad-1,or hTERT (reverse transcriptase component of the human telomerase, whichhas been shown to inhibit proliferation). Such genes are ectopicallyoverexpressed in the presence of the regulated oncogene, as describedwith Bcl-2 in the working examples herein. In addition, this aspect ofthe present invention includes using shRNA mediated gene knockdown (ordisruption or inhibition by any other method) for BH3-only members ofthe bcl-2 family that are proapoptotic (e.g., Bim, PUMA, NOXA, Bax, Bak,BclXS, Bad, Bar, and others), as well as disruption of Caspases 3, 9,10, MLL-1 (and all mammalian forms), Enl-1 (Endospermless-1) and allmammalian forms, Apaf-1 and other elements that form part of theapoptosome.

The nucleic acid sequence for each of these genes described above or thecoding region thereof is known in the art and is publicly available,including for humans. Similarly, the amino acid sequence for proteinsencoded by these genes is known in the art and is publicly available.

The present inventors have produced several different long-term,conditionally immortalized stem cells using the method of the presentinvention and using different combinations of protooncogenes andanti-apoptotic genes, including the following combinations: MYC-ER andBcl-2; MYC-ER and hTERT (reverse transcriptase component of the humantelomerase); ICN-1-ER and Bcl-2; ICN-1-ER and hTERT; and MYC-ER andICN-1-ER.

It is noted that with regard to either of the protooncogene or the geneencoding an anti-apoptosis protein used in the present method, it is notrequired that the entire gene be used in the constructs describedherein, since any portion of the gene or a nucleic acid sequence (e.g.,cDNA) that encodes the desired functional protein product, a functionalportion thereof, or a functional homologue thereof is encompassed by theinvention. Accordingly, reference generally herein to the genes ortransgenes used to transfect stem cells is to be understood to beexemplary and to include the use of any nucleic acid molecules encodingthe entire gene, the entire coding region of the gene, or portions ofthe genes or homologues thereof, as long as such nucleic acid sequencesencode functional proteins suitable for use in the present invention.

In one embodiment of the present invention, the present methodadditional includes the use of shRNAs or siRNAs that are directedagainst RNAs encoding proapoptotic proteins, such as the pro-apoptoticmembers of the Bcl-2 family, namely those of the BH3-only type (Bim,Bax, Bak, Puma, Noxa, etc.). The disruption of a pro-apoptotic gene inthe context of a regulated oncogene is expected to result in a moreefficient immortalization of certain stem cell populations. RNAinterference (RNAi) is a process whereby double stranded RNA, and inmammalian systems, short interfering RNA (siRNA) or short hairpin RNA(shRNA), is used to inhibit or silence expression of complementarygenes. In the target cell, siRNA are unwound and associate with an RNAinduced silencing complex (RISC), which is then guided to the mRNAsequences that are complementary to the siRNA, whereby the RISC cleavesthe mRNA. shRNA is transfected into a target cell in a vector where itis transcribed, and then processed by DICER enzymes to form siRNA-likemolecules that activate RISC, which, as with siRNA, is then guided tothe mRNA sequences that are complementary to the siRNA, whereby the RISCcleaves the mRNA.

The stem cells can be transfected with the vectors comprising theprotooncogene and encoding the anti-apoptosis protein using any suitablemethod of transfecting cells, and particularly mammalian cells,including by using combinations of techniques. The present inventorshave discovered that it is the particular coordination between the genes(or constructs) that are expressed that have resulted in the generationof conditionally immortalized, long term stem cells as described herein.The Examples have demonstrated the use of retroviral vectors, but othermethods include, but are not limited to, the use of other viruses andviral vectors derived therefrom, including, but not limited to,lentivirus vectors, parvovirus, vaccinia virus, coronavirus,calicivirus, papilloma virus, flavivirus, orthomixovirus, togavirus,picornavirus, adenoviral vectors, modified and attenuated herpesviruses.Any such virus can further be modified with specific surface expressedmolecules that target these to HSCs or other stem cells, such asmembrane bound SCF, or other stem-cell specific growth factor ligands.Other methods of transfection of mammalian cells include, but are notlimited to, direct electroporation of mammalian expression vectors, suchas by using NUCLEOFECTOR™ technology (AMAXA Biosystems). This technologyis a highly efficient non-viral gene transfer method for most primarycells and for hard-to-transfect cell lines, which is an improvement onthe long-known method of electroporation, based on the use of cell-typespecific combinations of electrical current and solutions to transferpolyanionic macromolecules directly into the nucleus. Additionally,suitable methods of transfection can include any bacterial, yeast orother artificial methods of gene delivery that are known in the art.

The step of expanding the transfected stem cells or culturing the stemcells and exogenous fusion proteins (e.g., the Tat-fusion proteinsdescribed in the variations of this method described below) in thepresence of suitable growth factors can include the use of any suitableculture conditions, including those specifically described herein. Thecombination of suitable stem cell growth factors can include any stemcell factors that allow transfected (e.g., transduced) cells of theinvention to grow, survive and proliferate in culture. While specificcombinations are described herein, and while this is an important stepof the present method, this step can be simply described as providingany combination of growth factors that are suitable for the growth,proliferation and survival of stem cells, and include any combinationsthat are known in the art. Accordingly, the invention is not limited toa particular combination. One preferred combination of growth factorsincludes: interleukin-6 (IL-6), IL-3 and stem cell factor (SCF). Anotherpreferred combination of growth factors includes stem cell factor (SCF),thrombopoietin (TPO), insulin-like Growth Factor 2 (IGF-2) andfibroblast Growth Factor 1 (FGF-1), in serum-free media. This lattercombination was recently described in Zhang and Lodich (2005; Murinehematopoietic stem cells change their surface phenotype during ex vivoexpansion, Blood 105, 4314-20). The stem cells transfected with nucleicacid molecules encoding the combinations proteins described herein(e.g., MYC-ER and Bcl-2 as described in the examples) are expected toalso become conditionally immortalized in this cocktail of growthfactors, as with the cocktail described in the Examples above (usingIL-3, IL-6 and SCF). Other growth factors for use in the inventioninclude, but are not limited to, angiopoietin-like proteins (e.g.,Agptl2, Angptl3, Angptl5, Angptl7, etc.), proliferin-2 (PLF2), glycogensynthase kinase-3 inhibitors, inducers of the wnt and Notch signalingpathways, Flt3L and related cytokines, fibroblast growth factor 2 (FGF2)and related cytokines, writ-1 and other activators of the Wnt pathway,Sonic hedghog (shh-1) and other activators of that pathway. Othersuitable combinations of growth factors will be applicable to the methodof the present invention and will be apparent to those of skill in theart. Indeed, the cell lines generated using the method of the presentinvention can readily be used to screen for additional cytokines andgrowth factors that could be used for expanding long-term stem cells, orany of their derived progenitors, in vitro under neutral or directedconditions.

According to the present invention, a medium suitable for culture ofanimal cells can include any available medium which has been developedfor culture of animal cells and particularly, mammalian cells, or whichcan be prepared in the laboratory with the appropriate componentsnecessary for animal cell growth, such as assimilable carbon, nitrogenand micronutrients. Such a medium comprises a base medium, which is anybase medium suitable for animal cell growth, including, but not limitedto, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's modifiedEagles medium (DMEM), alpha MEM (Gibco), RPMI 1640, or any othersuitable commercially available media. To the base medium, assimilablesources of carbon, nitrogen and micro-nutrients are added including, butnot limited to, a serum source, growth factors, amino acids,antibiotics, vitamins, reducing agents, and/or sugar sources. It isnoted that completed mediums comprising a base medium and many of theadditional components necessary for animal cell growth are commerciallyavailable, and some media are available for particular types of cellculture. In addition, many serum-free media are available and may beparticularly suited for the culture of stem cells according to theinvention.

Cells and Compositions

Another embodiment of the present invention relates to a cell, cellline, or population of cells produced according to the method of thepresent invention as described herein. Also included in the inventionare compositions comprising such cells, cell lines or populations ofcells. For therapeutic methods, such compositions can include apharmaceutically acceptable carrier, which includes pharmaceuticallyacceptable excipients and/or delivery vehicles, for delivering thecells, cell lines, or cell populations to a patient. As used herein, apharmaceutically acceptable carrier refers to any substance suitable fordelivering a therapeutic composition useful in the method of the presentinvention to a suitable in vivo site.

Adaptation of the Method of the Invention to Produce Cell Lineages atIntermediate Stages of Development

Another embodiment of the present invention relates to adaptations ofthe novel methods described herein to generate cell lines that captureintermediate stages of development for the hematopoietic lineages.According to the present invention, an “intermediate” stage ofdevelopment or differentiation refers to a pluripotent stage of celldevelopment or differentiation that is downstream of the stage ofdevelopment or differentiation of the stem cell from which the“intermediate” cell was derived, but is upstream of the final, orterminal, point of differentiation of a cell. For example, a pre-B cellis an intermediate stage of a hematopoietic stem cell, which can stilldifferentiate into a mature B cell. Intermediate stages of developmentor differentiation will be understood by those of skill in the art.

More particularly, for many therapeutic and discovery or researchapplications, as well as for storage of cells lines, it is desirablethat the cell lines have a stable phenotype and retain their ability tofurther differentiate along their committed pathway once the activeoncogene with which the cell has been transfected is turned off.Accordingly, the present invention encompasses additional steps ofproducing cells that have not fully differentiated (are not terminallydifferentiated), but rather, are at an intermediate stage ofdifferentiation. In one non-limiting example of this embodiment,long-term stem cells-produced using the method described above arerandomly differentiated in vitro following withdrawal of the conditionsthat maintain the activity of the protooncogene or other gene thatpromotes cell survival and proliferation (e.g., 4-OHT in the case of thetamoxifen-dependent protooncogenes), or by applying the appropriateconditions that turn off (inactivate) the protooncogene/oncogene. Thisstep can be performed while maintaining the culture in neutral cytokinegrowth conditions (e.g., IL-3, IL-6 and SCF), or by replacing thosecytokines which could specifically direct differentiation towards acertain lineage (e.g., IL-7 and Notch ligands for lymphoid lineages,GM-CSF and IL-4 for dendritic cells, G-CSF for myelomonocytic cells,etc.) with cytokines that are neutral for differentiation (do not director drive differentiation of the cells). Once the cultures begin todisplay differentiation markers consistent with a specific lineage, theculture media is again supplemented with the conditions that activatethe protooncogene (e.g., 4-OHT) or exposed to the conditions thatotherwise reactivating the protooncogene, in order to stabilize thephenotype and generate cell lines having a stable, intermediatedifferentiation phenotype.

By way of exemplification of this method, the inventors have generatedCD4+, αβ+ T cells in vitro from ABM42 cells (lt-HSC produced by themethod of the invention; see Examples) by withdrawal of 4-OHT from themedia, and re-addition of 4OHT after differentiation. The inventors havealso generated dendritic cell lines by incubating ABM46 cells (seeExamples) in GM-CSF, IL-4 and FLT3L and then placing the cultures backin the presence of 4-OHT after differentiation.

Another approach for creating such cell lines involves introducing thectlt-HSC cells into mice to allow for differentiation, and arresting, orstabilizing the phenotypes in vivo after injections of 4-OHT. Thismethod is described in detail in Example 8. Briefly, and by way ofexample, lt-HSC generated by the present method are injected intoimmuno-compromised animals (e.g., immuno-compromised mice). The oncogenein the lt-HSCs is reactivated using injections of the activating agent(e.g., 4-OHT), cells are later collected, and then the cells can becultured in vitro to differentiate the cells, and then stored or used asdesired. This approach, and the other described above, can be used forboth murine and human ctlt-HSC cell lines, such as by using eitherNOD/SCID mice as the recipients, or neonatal Rag-1^(−/−) mice, whichwill be given intrahepatic injections.

Application of the Method of the Invention to Embryonic Stem Cells

Another embodiment of the invention relates to the application of themethod of conditionally immortalizing stem cells to embryonic stem (ES)cells. Such methods will be useful for generating cell lines that aremore readily derived from ES cells, such as cells of the neuronallinage, including neuronal stem cells.

In this embodiment, the method of the present invention, comprising thetransduction of cells (in this case, ES cells) with a protooncogene anda gene that inhibits apoptosis (e.g., MYC-ER and Bcl-2) can be appliedto ES cells to further control the directed differentiation of thesecells. In this embodiment, such cells can be used to generate transgenicmice, for example, and in addition, any ES cell and relevant progenitorcell population derived therefrom can be subjected to the activation ofthe protooncogene by exposure to the activating agent, hence allowingfor the generation of novel conditionally transformed stem cell lines(different tissue types), or mature cell lines for the tissue type ofinterest. In addition, the directed differentiation of transduced EScells in vitro can also be used to capture intermediate states ofdifferentiation by as described above. The use of ES cells or ES-derivedcells in this manner provides a novel platform for drug discovery andtarget identification in the setting of different diseases.

For example, neuronal stem cells can be employed in this embodiment ofthe invention, as well as the directed differentiation of ES cells intothe neuronal pathway using the method of the invention. The isolationand transduction of neuronal stem cells from the hippocampus has beenpreviously described for mice. The culture conditions for neurosphereswould enable the proliferation of those cells, rendering themsusceptible to viral-mediated transduction of the genes of the invention(e.g., MYC-ER and Bcl-2), in order to generate conditionally transformedneuronal stem cell lines. Their differentiation in vitro as well as invivo following implantation can be monitored by virtue of the virallyencoded reporter genes as well as previously defined markers of neuronaldifferentiation. In addition, the administration of the activating agent(e.g., 4-OHT) to the mice following transplantation of the conditionallytransformed neuronal stem cell lines may lead to the development of aneurological malignancy (neuroblastoma, glioblastoma, etc.). Thosetumors would provide a novel model for preclinical studies and targetidentification.

The directed differentiation of ES cells that had been transduced with,for example, MYC-ER and Bcl-2, can be carried out in the presence of apreviously defined growth medium, as well as cytokines. The addition ofthe activating agent (e.g., 4-OHT) at any time during the culture willenable the stabilization of the cells at an intermediate phenotype, andleads to the generation of cell lines that still retain the capacity toundergo further differentiation. For instance, the generation ofdopaminergic neurons from ES cells is normally done by the addition ofRetinoic acid and FGF8. This type of neuron would be ideal for repairingbrain lesions observed in Alzheimer's patients. However, thetransplantation of fully differentiated neuronal cells may precludetheir successful implantation and engraftment. A conditionallytransformed cell line that was committed to the dopaminergic neuronalpathway, but still retained its ability to further differentiate aftertransplant, as envisioned herein, is expected to greatly increase thechances of implantation and successful engraftment. A similar scenariocan be proposed for the generation of motor neurons from ES cells, byadding Retinoic acid and a sonic hedgehog agonist to the cultures. Thoseneuronal cells could help repair spinal cord injuries. Once again, fullydifferentiated cells would not be used in this embodiment, but rather,the committed progenitor cells that retain the capacity to differentiate(produced by the method of the invention) would be employed.

Variations or Modifications of the Method of Conditional Immortalizationfor the Removal of the Transgene

In one embodiment of the invention, in order to avoid taking the risk ofintroducing stem cells that harbor transgenes such as those describedherein (e.g., MYC-ER) into humans and/or mice, the recombinantconstructs are designed so that these DNA fragments will be excised.This embodiment can be achieved using any suitable method of firstestablishing the long-term stem cells according to the method of theinvention, and then exposing the cells (or a patient) to conditionsunder which the recombinant DNA will be removed, excised or completelysilenced.

For example, in one aspect of the invention, a bacterial recombinaseapproach is used. In this aspect of the invention, preferably, twodifferent recombinases are used in order to allow control over which oneof the two genes is excised at any one point in time. Two examples ofsuch recombinases are the Cre and Flp recombinases, which are well-knownin the art. Briefly, the recognition substrate sequences (RSS's) for oneof the recombinases is introduced into the retroviral constructs suchthat they flank the open reading frame of the oncogene, as well as thereporter gene (e.g., GFP or Thy1.1). In this case, the cells areincubated in media containing a Tat-Cre fusion protein (i.e., HIV orother retroviral Tat protein fused to Cre). This recombinant protein hasbeen previously described and shown to be able to passively enter cells,and mediate loxP site-dependent recombination of genomic DNA. Othermethods of gene (nucleic acid molecule) excision are known to those ofskill in the art and could readily be applied to the present invention.Examples 5 and 13 exemplify this embodiment of the invention.

In another embodiment of the invention, to provide another method ofavoiding the risk of introducing stem cells that harbor transgenes suchas those described herein into humans and/or other animals (e.g., mice),instead of transfecting the stem cells with the combination of therecombinant constructs for the protooncogene or the anti-apoptosisprotein, the invention is performed by making use of Tat-fusion proteinsas a method to allow the proteins access to the inside of the cellwithout having to introduce transgenes into the cell. For example,recombinant constructs that encode tat-protooncogene ortat-anti-apoptosis genes (e.g., Tat-MYC-ER or Tat-Bcl-2) may be used toconditionally immortalize stem cells. In this embodiment of theinvention, the target stern cells will be cultured under suitableculture conditions, in media that contains purified recombinantTat-fusion proteins encoded by the specific gene combination selected(e.g., MYC-ER and Bcl-2). In this embodiment of the invention, theprotooncogene product or similar gene product can be inducible, as inthe embodiments above. Alternatively, or in addition, the action of thisprotein can be regulated simply by providing or removing the proteinfrom the culture. While the cell lines that are generated with thisapproach will be continuously dependent upon the addition of theexogenous Tat-fusion proteins, they will not have a specific exogenousnucleotide sequence introduced into them. The absence of foreignoncogene sequences is expected to improve the clinical deployment of themethod of the present invention. Human immunodeficiency virus-1 (HIV-1)Tat, is one exemplary Tat protein, although other retroviral Tatproteins are known in the art. As a non-limiting example, the nucleicacid sequence encoding HIV-1 Tat is represented herein as SEQ ID NO:9,which encodes an amino acid sequence represented herein by SEQ ID NO:10.

In another embodiment, to provide another method of avoiding the risk ofintroducing stem cells that harbor transgenes such as those describedherein into humans and/or other animals (e.g., mice), instead oftransfecting the stem cells with the combination of the recombinantconstructs for the protooncogene or the anti-apoptosis protein, theinvention is performed by introducing proteins (e.g., MYC and Bcl-2)into a cell using aptamer technology. Aptamers are short strands ofsynthetic nucleic acids (usually RNA but also DNA) selected fromrandomized combinatorial nucleic acid libraries by virtue of theirability to bind to a predetermined specific target molecule with highaffinity and specificity. Aptamers assume a defined three-dimensionalstructure and are capable of discriminating between compounds with verysmall differences in structure. Accordingly aptamers can be conjugatedwith the proteins used in the invention or with non-integrating cDNAencoding the proteins, for example, and used to deliver the proteins orDNA to the cells. In addition, aptamers can readily be used to deliversiRNA to cells, for example, when one disrupts proapoptotic proteinsaccording to the present invention. Aptamer technology is discussed, forexample, in Davidson, 2006, Nature Biotechnol. 24(8):951-952; andMcNamara et al., 2006, Nature Biotechnol. 24(8):1005-1015). Again, theabsence of foreign oncogene sequences is expected to improve theclinical deployment of the method of the present invention.

In another embodiment, to provide another method of avoiding the risk ofintroducing stem cells that harbor transgenes such as those describedherein into humans and/or other animals (e.g., mice), instead oftransfecting the stem cells with the combination of the recombinantconstructs for the protooncogene or the anti-apoptosis protein, theinvention is performed by introducing the protooncogene and/oranti-apoptosis protein into a cell using CHARIOT™ technology (KrackelerScientific, Inc, Albany, N.Y.). With this technology, a non-covalentbond is formed between a CHARIOT™ peptide and the protein of interest.This protects the protein from degradation and preserves its naturalcharacteristics during the transfection process. Upon delivery to acell, the complex dissociates and CHARIOT™ is transported to thenucleus, while the delivered protein is biologically active and free toproceed to its cellular target. Efficient delivery can occur in thepresence or absence of serum, and is independent of the endosomalpathway, which can modify macromolecules during internalization. Thisdelivery system also bypasses the transcription-translation process.Accordingly, the proteins useful in the present invention can bedelivered to a cell and released to conditionally immortalize the cell,without the need for the introduction of a protooncogene or oncogenes tothe cell. As above, the absence of foreign oncogene sequences isexpected to improve the clinical deployment of the method of the presentinvention.

As yet another alternative (or additional) means to control for thepossibility of an insertion of a protooncogene into the host cell genomeby the various viral approaches described herein, and thereby avoid atransforming event, a drug sensitivity (drug susceptibility) cassettecan be introduced into the viral constructs to be used such that it willbe expressed in every transduced cell and its differentiated progeny. Adrug sensitivity cassette or a drug susceptibility cassette is a nucleicacid sequence encoding a protein that renders a cell susceptible orsensitive to the presence of a particular drug, so that upon exposure tothe drug, the cell activity is inhibited and preferably, undergoesapoptosis. Those patients in which the levels of a particular blood cellpopulation increases without apparent cause (e.g., infection, trauma,stress, etc.), can be given a course of the drug to which sensitivityhas been introduced in order to ablate those cells and mitigate anypossible additional complications involving cells in which the geneticinsertions may have inadvertently caused an oncogenic mutation.Accordingly, as a non-limiting example, one could introduce into aconstruct used in the method of the invention a cassette that encodesthe cDNA for HPRT in order to render the transduced cells susceptible to6-thioguanine. Another non-limiting example is the introduction of thethymidine kinase cDNA from a Herpes-simplex virus family member(HSV-TK), in order to render the transduced cells susceptible torelevant inhibitors such as Ganciclovir, Acyclovir, and any relevantderivatives. In addition, any other such drug sensitivity cassettes andtheir relevant agonists would work in this context.

Other methods of introducing nucleic acids or proteins according to thepresent invention into a cell will be apparent to those of skill in theart. Those that minimize or eliminate the risk of introducingrecombinant DNA into a host cell genome are preferred by the invention,many such examples being described above.

Methods of Use for Conditionally Immortalized Cells of the Invention

Another embodiment of the present invention includes any of the stemcell populations, including mixed and clonal populations, that areproduced by the method of the invention, as well as the use of the stemcells of the invention in any of the methods described herein, includingdifferentiation into a desired cell type, and any method oftransplantation, cell replacement, disease therapy, genetic engineering,drug discovery, and investigation of cell development anddifferentiation as described herein.

Since one can now produce virtually unlimited supplies of homogeneousstem cells that can readily be stored, recovered, expanded andmanipulated, such stem cells can be used as stem cells or differentiatedinto various cell lineages and used in assays to test various compoundsfor effects on cell differentiation, gene expression, and cellprocesses. Therefore, one embodiment of the invention relates to amethod to identify compounds that effect cell differentiation, geneexpression, and/or cell processes. The method generally includes thesteps of contacting stem cells produced by the method of the presentinvention with a compound to be tested, and measuring a particularresult, and particularly a desired result, such as gene expression, abiological activity, cell differentiation, cell growth, cellproliferation, etc. (see below), as compared to in the absence of thecompound, to determine whether or not the test compound had the desiredeffect on the stem cell. This method can be used to test for virtuallyany aspect of cell differentiation, cell activity or gene expression. Inone aspect, the stem cells are manipulated prior to contact with thecompounds, such as by genetic manipulation. Stem cells from individualswith genetic defects can be evaluated in such assays in order toidentify therapeutic compounds (e.g., cancer therapeutics) and toevaluate gene replacement therapies, for example. Indeed, the technologyof the present invention provides an opportunity to target the cells ofa specific individual to identify drug candidates and therapeuticcandidates and strategies that are “tailored” to the cells of anindividual. Furthermore, as discussed above, such assays can also beused to identify other growth factors or culture conditions that aresuitable for maintaining the stem cells of the invention in culture. Anexample of such an assay is described in detail below in Example 7,although the invention is not limited to this assay.

Another embodiment of the invention relates to a method to study celllineage commitment and/or differentiation and development of cells froma stem cell, which generally comprises culturing the conditionallyimmortalized stem cells of the present invention and evaluating suchcells for genetic and biological markers related to cell development anddifferentiation under various conditions and in the presence and absenceof compounds or agents that may affect cell lineage commitment ordifferentiation. As discussed above, prior to the present invention,such studies were severely hampered by the lack of access to and theinability to generate sufficient numbers of the desired cell populationto perform desired experiments. For example, in order to identify orscreen for intermediates in the differentiation of a particularprogenitor cell line, a sufficient number of cells must be obtained toprovide meaningful and reproducible results. Using technologiesavailable at the time of the invention, this was not possible. However,the present invention solves the problem by providing expandable andessentially unlimited supplies of homogeneous stem cells that can beused in a variety of experiments. This technology will greatly enhanceresearch capabilities in the area of cell differentiation and discovery.In one aspect, conditionally immortalized stem cells of the inventionare expanded, and then a subset are cultured in the absence of theconditions that maintain the cells in the conditionally immortalizedstate (e.g., in the absence of tamoxifen, according to the exemplarymethod illustrated herein). The cells can be evaluated for changes ingene expression, cell surface markers, secretion of biomolecules, or anyother genotypic or phenotypic marker, to study the process of celldifferentiation and lineage commitment. Growth factors or other factorscan be added to the cultures, for example to drive differentiation downa particular cell lineage pathway, and the changes in the cells can beevaluated in the presence or absence of such factors. Furthermore, thecells can be used to evaluate culture conditions, in vivo conditions,factors, and agents that influence (regulate) cell differentiation anddevelopment.

Various methods of detection of changes in genotypic or phenotypiccharacteristics of cells in any of the assays of the invention are knownin the art. Examples of methods that can be used to measure or detectgene sequence or expression include, but are not limited to, polymerasechain reaction (PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR,quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northernblot, sequence analysis, microarray analysis, detection of a reportergene, or other DNA/RNA hybridization platforms. Methods to measureprotein levels, include, but are not limited to: Western blot,immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay(RIA), immunoprecipitation, surface plasmon resonance,chemiluminescence, fluorescent polarization, phosphorescence,immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,microcytometry, microarray, microscopy, fluorescence activated cellsorting (FACS), flow cytometry, and assays based on a property of theprotein including but not limited to DNA binding, ligand binding,interaction with other protein partners, cell signal transduction,enzyme activity, and secretion of soluble factors or proteins.

In drug screening assays, the term “test compound”, “putative inhibitorycompound” or “putative regulatory compound” refers to compounds havingan unknown or previously unappreciated regulatory activity in aparticular process. As such, the term “identify” with regard to methodsto identify compounds is intended to include all compounds, theusefulness of which as a compound for a particular purpose (e.g.,regulation of cell differentiation) is determined by a method of thepresent invention, preferably in the presence and absence of such acompound. Compounds to be screened in the methods of the inventioninclude known organic compounds such as antibodies, products of peptidelibraries, and products of chemical combinatorial libraries. Compoundsmay also be identified using rational drug design. Such methods areknown to those of skill in the art and can involve the use ofthree-dimensional imaging software programs. For example, variousmethods of drug design, useful to design or select mimetics or othertherapeutic compounds useful in the present invention are disclosed inMaulik et al., 1997, Molecular Biotechnology: Therapeutic Applicationsand Strategies, Wiley-Liss, Inc., which is incorporated herein byreference in its entirety.

In any of the above-described assays, the conditions under which a cell,cell lysate, nucleic acid molecule or protein of the present inventionis exposed to or contacted with a putative regulatory compound, such asby mixing, are any suitable culture or assay conditions, which caninclude the use of an effective medium in which the cell can be cultured(e.g., as described above) or in which the cell lysate can be evaluatedin the presence and absence of a putative regulatory compound. Cells ofthe present invention can be cultured in a variety of containersincluding, but not limited to, tissue culture flasks, test tubes,microtiter dishes, and petri plates. Culturing is carried out at atemperature, pH and carbon dioxide content appropriate for the cell.Such culturing conditions are also within the skill in the art, andparticularly suitable conditions for culturing conditionallyimmortalized stem cells of the present invention are described in detailelsewhere herein. Cells are contacted with a putative regulatorycompound under conditions which take into account the number of cellsper container contacted, the concentration of putative regulatorycompound(s) administered to a cell, the incubation time of the putativeregulatory compound with the cell, and the concentration of compoundadministered to a cell. Determination of effective protocols can beaccomplished by those skilled in the art based on variables such as thesize of the container, the volume of liquid in the container, conditionsknown to be suitable for the culture of the particular cell type used inthe assay, and the chemical composition of the putative regulatorycompound (i.e., size, charge etc.) being tested.

In one embodiment of the invention, the cells and methods of theinvention are useful for methods directed at evaluating pluripotency ofctlt-HSCs derived from human cord blood, CD34+ cells, or adult CD34+cells isolated from peripheral blood. Such a method is described inExample 11.

Yet another embodiment of the invention relates to the use of tilt-HSCcell lines as a platform to generate novel models of Acute MyeloidLeukemia (AML). More particularly, the present inventors have generateda mouse model of acute myeloid leukemia using the ctlt-HSCs of theinvention. These are leukemias composed of cells that resemble HSCs,based on their surface marker expression. In order to generate ctlt-HSCsto promote leukemia in mice, 10³-10⁵ ctlt-HSCs are transferred alongwith 10⁵ Rag-1^(−/−) whole bone marrow cells into lethally irradiatedrecipient mice. The mice are given weekly doses of 4-OHT in order tomaintain oncogene activity, and monitored for clinical signs associatedwith leukemia, as known in the art. Tumors have been recovered fromthese animals and they can be propagated in culture in the absence of4-OHT. Those cells retain their HSC-like phenotype, indicating that theyare no longer exquisitely dependent upon MYC hyperactivity in order forproliferation, survival and arrested differentiation. The leukemic celllines can also confer the disease upon secondary transplantation toirradiated recipient mice. These tools provide a novel platform forstudying the biology and exporting new therapeutic avenues for AML andrelated diseases. Furthermore, the introduction of ctlt-HSC cell linesinto mice that are treated with 4-OHT will provide a good built-inpositive control for therapy: the withdrawal of 4-OHT. The secondarycell lines that arose after the establishment of tumors in vivo can alsobe used to understand the relevant therapeutic targets for drugresistant forms of AML.

Other embodiments of the present invention relate to the use of the stemcells generated by the method of the present invention, as well as cellsdifferentiated from those stem cells, in a variety of therapeutic andhealth-related methods. These methods generally include the steps ofobtaining a population, culture or line of conditionally immortalizedstem cells produced by the method of the present invention, removing theconditions under which such cells are conditionally immortalized, andthen using the cells in a therapeutic protocol. For example, the cellscan be administered directly to an individual in need of the cells orthe cells can be differentiated into a desired cell type in vitro andthen administered to an individual. In addition, prior to or just afterthe removal of the conditions under which the cells are immortalized,the cells can be genetically modified in vitro to express or silence agene or genes, as a novel method of gene therapy under a controlledenvironment. The cells can then be administered to an individual as stemcells or first differentiated in vitro to a desired cell lineage.

To obtain the stem cells, in one embodiment, stem cells are obtainedfrom the individual to be treated, and are then conditionallyimmortalized according to the method of the invention. These cells canbe expanded extensively, stored (e.g., frozen or cryopreserved), andthen retrieved and expanded again, manipulated, and/or used repeatedlyas required. In another embodiment, one obtains the stem cells byaccessing a previously stored source of conditionally immortalized stemcells from the individual to be treated. In yet another embodiment, thestem cells are obtained from a panel of human stem cell lines that werepreviously generated and which cover a significant percentage of thepopulation according to the current criteria used to identify “matching”donors. In one embodiment, the cells are obtained from fresh, orcryopreserved cord blood, hematopoietic progenitor populations that canbe derived from the directed differentiation of ES cells in vitro, HSCsobtained from the peripheral blood of normal, or G-CSF treated patientswho have been induced to mobilize their lt-HSCs to the peripheralcirculation. Other sources of stem cells will be apparent to those ofskill in the art. The cells are cultured according to the methodsdescribed previously herein and the conditions controllingimmortalization can be removed at the appropriate time. In addition,prior to administration of the cells to an individual, the cells can bemanipulated to excise the genes or constructs that are responsible forthe conditional immortalization (i.e., the protooncogene and/or theanti-apoptosis encoding gene), or if the cells are maintained throughthe use of soluble fusion proteins in the culture medium, as describedabove for the Tat-fusions, these soluble proteins can be removed fromthe culture gradually or immediately.

Therefore, the present invention includes the delivery of stem cellsproduced by the method of the invention (including compositionscomprising such stem cells), or cells differentiated from these cells,to an individual (which can include any animal). Since the stem cellsused in these methods are produced in vitro, even if stem cells wereinitially isolated from the patient, the entire administration processof the cells is essentially an ex vivo administration protocol. Ex vivoadministration refers to performing part of the regulatory step outsideof the patient, such producing the conditionally immortalized stem cellsthat were removed from an individual (which can include producinggenetically modified stem cells in addition to essentially normal stemcells), and returning the cells, or cells differentiated from thesecells, to the patient. The stem cells produced according to the presentinvention or cells differentiated therefrom can be returned to anindividual, or administered to an individual, by any suitable mode ofadministration. Such administration can be systemic, mucosal and/orproximal to the location of a target site. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated or the reason foradministration. Preferred methods of administration include, but are notlimited to, intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,intraspinal, pulmonary administration, impregnation of a catheter, anddirect injection into a tissue (e.g., such as cannulation of the liver,for example).

The cells can be administered with carriers or pharmaceuticallyacceptable excipients. Carriers are typically compounds that increasethe half-life of a therapeutic composition in the treated individual.Suitable carriers include, but are not limited to, polymeric controlledrelease formulations, biodegradable implants, liposomes, oils, esters,and glycols. As used herein, a pharmaceutically acceptable excipientrefers to any substance suitable for delivering cells produced by themethod of the present invention to a suitable in vivo site. Preferredpharmaceutically acceptable excipients are capable of maintaining acells in a form that, upon arrival of the cells at a target tissue orsite in the body, the cells are capable of functioning in a manner thatis beneficial to the individual.

According to the present invention, an effective administration protocolcomprises suitable dose parameters and modes of administration thatresult in delivery of a useful number of functional cells to a patientin order to provide a transient or long-term benefit to the patient.Effective dose parameters can be determined using methods standard inthe art for a particular condition or disease. Such methods include, forexample, determination of survival rates, side effects (i.e., toxicity)and progression or regression of disease.

A suitable single dose of stem cells or cells differentiated therefromaccording to the present invention is a dose that is capable ofproviding a beneficial number of cells to a patient, when administeredone or more times over a suitable time period. For example, a preferredsingle dose of stem cells according to the present invention is fromabout 0.5×10⁴ to about 5.5×10⁸, or from about 0.5×10⁵ to about 5.5×10⁷,or from about 0.5×10⁶ to about 5.5×10¹⁰ stem cells per individual peradministration, with doses from about 1×10⁸ to about 5.5×10¹⁰ being evenmore preferred. Any dose in between 0.5×10⁴ and about 5.5×10¹⁰ isencompassed by the invention, in increments of 10² cells. Higher orlower doses will be known to those of skill in the art depending on thetype of stem cell or differentiated cell to be administered, and alsodepending on the route of administration. It will be obvious to one ofskill in the art that the number of doses administered to an animal isdependent upon the extent of the condition or disease and the responseof an individual patient to the treatment. Thus, it is within the scopeof the present invention that a suitable number of doses includes anynumber required to treat a given disease.

As used herein, the phrase “protected from a disease” refers to reducingthe symptoms of the disease; reducing the occurrence of the disease,and/or reducing the severity of the disease. Protecting an animal (anindividual, a subject) can refer to the ability of cells producedaccording to the present invention, when administered to an animal, toprevent a disease from occurring and/or to cure or to alleviate diseasesymptoms, signs or causes. As such, to protect an animal from a diseaseincludes both preventing disease occurrence (prophylactic treatment) andtreating an animal that has a disease or that is experiencing initialsymptoms of a disease (therapeutic treatment). The term, “disease”refers to any deviation from the normal health of a mammal and includesa state when disease symptoms are present, as well as conditions inwhich a deviation (e.g., infection, gene mutation, genetic defect, etc.)has occurred, but symptoms are not yet manifested.

As discussed above, the stem cells of the present invention can beadministered to an individual to treat or prevent a variety ofconditions. For example, the stem cell lines of the present inventionprovide a unique source of expandable stem cells for use in a variety oftransplantation and therapeutic strategies, including the treatment ofcancer, and particularly, cancer that is treated by radiation. Inaddition, a variety of immune deficiency disorders and anemia disorders(e.g., aplastic anemia or hemolytic anemia) will also benefit greatlyfrom this technology, since the present invention provides the abilityto repopulate hematopoietic cells of an individual as needed by theindividual. Another application of the present invention relates to thegeneration of continuously expandable and renewable hair follicle stemcells, for use, for example in the context of reconstructive surgery forburn victims, for any individual that undergoes chemotherapy and/orradiation therapy resulting in the irreversible loss of hair growth, aswell as patients following any surgical procedure affecting the skull orin elective procedures that involve the induction of hair growth inindividuals affected by hereditary pattern baldness. Similarly,application of the present invention to stem cells of the skin will beinvaluable for use in wound healing and treatment of burn victims, aswell as plastic reconstructive surgery for trauma and other patients, aswell as elective surgeries, including, but not limited to, cosmeticsurgery. Such cells can be additionally genetically manipulated tocorrect inborn or acquired genetic defects in young and agedindividuals. One of skill in the art will understand based on thisdisclosure that benefits can be derived from the use of the presentinvention on various other stem cell populations, including, but notlimited to, stem cells derived from lung, breast, and intestinalepithelium and stem cells derived from neural and cardiac tissue, toname just a few.

In addition, as discussed above, the present invention provides theunique opportunity for an individual to have access to expandablesupplies of autologous stem cells and cells differentiated therefrom asneeded throughout the life of the individual. Such stem cells generatedby the present method can be stored and used as part of therapeuticprotocols during the lifetime of the individual, should they be needed(e.g., in the event the individual develops a cancer or immunedeficiency disease).

Genetic defects can now be corrected or beneficial gene modificationscan be introduced into somatic cells by manipulating autologous stemcells obtained from an individual that have been conditionallyimmortalized and expanded using the method of the present invention. Thestem cells can then be reintroduced into the individual from whom theywere obtained.

Additional applications of the present invention include the use of stemcell lines to repair lung injury that occurs as a result of COPD, IPF,emphysema, asthma and smoking. In addition, such cells could be used totreat blood vessel damage in the heart, and help in autoimmune diseasesafter lethal irradiation (e.g., SLE, diabetes, RA).

In the method of the present invention, cells produced according to themethod of the invention and compositions comprising the cells can beadministered to any animal, including any member of the Vertebrateclass, Mammalia, including, without limitation, primates, rodents,livestock and domestic pets. A preferred mammal to treat is a human.

Various aspects of the present invention are described in more detail inthe following Examples and the attached figures. However, the presentinvention is not limited to these examples and illustrations of theinvention.

EXAMPLES Example 1

The following example describes the development of a method toreversibly immortalize long-term hematopoietic stem cells (lt-HSCs).

Elucidation of the molecular basis of the impairment in hematopoieticlineage development has been complicated historically by the lowfrequency of relevant cell populations, which prevents biochemicalanalysis of signaling and downstream responses. In fact, this has been amajor limiting factor in all studies of hematopoiesis. In addition, thelimited availability of LT-HSCs has also been a major obstacle in thetreatment of many types of cancer as well as several kinds of immunedeficiencies in humans.

In an effort to overcome this limitation, the present inventorsdeveloped a method to produce conditionally transformed cell linesrepresenting early hematopoietic stem cell progenitors. The initialstrategy involved retroviral transduction of bone marrow stem cells from5FU treated young and immunologically aged 3-83 mice. The inventorsutilized the pMSCV bisistronic retroviral vector with inserts encodingBcl-2 and GFP, and MYC-ER and GFP [Van Parijs, L., Y. Refaeli, A. K.Abbas, and D. Baltimore. (1999) Autoimmunity as a consequence ofretrovirus-mediated expression of C-FLIP in lymphocytes. Immunity, 11,763-70]. These genes were selected because the present inventors knewthat MYC has the ability to replace cytokine derived survival andproliferative signals in lymphocytes. By restricting the target cell,the inventors hypothesized that stem cell tumors might form.Importantly, MYC-ER function is tamoxifen dependent in this setting,allowing the termination of MYC function and transformation bywithdrawing tamoxifen from the animal or cultures. In cells transducedwith MYC-ER, the fusion protein is produced, but is retained in thecytoplasm until exposed to tamoxifen.

More specifically, stem cell populations from 5FU treated mice weretransduced with both retroviruses (encoding MYC-ER and Bcl-2) andtransferred into lethally irradiated recipient mice (1200 rads). Tendays later, weekly intraperitoneal injections of 1 mg/mouse of4-hydroxytamoxifen (4OHT) emulsified in oil were initiated to activateMYC function (FIG. 1). Within four weeks, recipients of young (but notold) transduced stem cells developed tumors. The tumors were harvestedfrom bone marrow, spleen and lymph nodes and cultured in vitro withtamoxifen, but without added cytokines. These cells grew for about 10days, but then growth stopped and the cells eventually died, theinventors suspected that the cells were differentiating and consideredthat this might have been due to requirements for cytokines for growthof the cells. Referring to FIG. 1, the curves represent the kinetics ofmortality after transplantation and activation of MYC function in vivo.The mice uniformly succumbed to leukemias. While the overexpression ofMYC can replace the cytokine-dependent proliferation and survivalfunction, it does not seem to be involved in the cytokine-deriveddifferentiation signals.

When ill, the mice were euthanized. Bone marrow, spleen and lymph nodecells were harvested and placed in culture with tamoxifen and a stemcell growth factor cocktail (IL-6, IL-3 and stem cell factor (SCF)). Inparallel, cells were analyzed by flow cytometry (FIG. 2). Referring toFIG. 2, the dot plots represent the flow cytometric data for the forward(FSC) and side (SSC) scatter characteristics of the HSCs after threedays in culture with IL-3, IL-6 and SCF. These two criteria correlatewith cell size (FSC) and granularity (SSC). The two populations havesimilar profiles. The histograms represent the levels of GFP expressedin each cell population. This reflects the efficiency of retroviraltransduction in vitro with retroviruses that encode cDNAs for MYC-ER andBcl-2.

In all cases, ex vivo GFP cells were >90% Sca-1⁺ and Lineage markernegative. After a few days in culture, cells began to grow andapproximately 400 lines were frozen for later study. After propagation,these cells retained expression of EGFP and were homogeneously positivefor SCA1 and negative for CD34, Flk2 and lineage markers (FIG. 3). Theonly difference in marker expression between young mouse-derived andaged mouse-derived markers was increased expression of c-kit in young.Without being bound by theory, the present inventors believe that thismay have resulted from longer culture (3 months vs. 3 weeks) of agedlines in c-kit ligand before markers were analyzed. Finally, theinventors discovered that these lines can be recovered easily afterfreezing and retained their original phenotype. Importantly, these celllines are homogenous in phenotype and exhibit the phenotype of lt-HSCthat provide all long term reconstitution in mice (Reya, T., Duncan, A.W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L.,Nusse, R., and Weissman, I. L. (2003). A role for Wnt signaling inself-renewal of hematopoietic stem cells. Nature 423, 409-14).

Recently, the inventors thawed 10 bone marrow derived lines produced asdescribed above, and were able to recover 9 our of 10 of these lineseasily by culture in the cytokine cocktail and 4OHT. The inventorsphenotyped these tumors, and the results were extremely promising.Specifically, each line contained two distinct cell populations based onforward and 90° light scatter. The nine lines differed only in theproportionality of these populations. The larger of these populations incell size were uniformly GFP bright and positive for Sca1, Endoglin andckit but negative for Flt3, B220, CD19 and mIgM. CD34⁻ also appeared tobe negative, although this required confirmation (FIG. 3). Thisphenotype corresponds perfectly with the published characteristics oflong term repopulating pluripotent stem cells (Reya et al., supra). Theinventors observed the same initial phenotype on the cell lines thatthey recently obtained from leukemias that developed from transducedHSCs obtained from young donor mice (FIG. 3).

To test the ability of these cells to differentiate, representativelines were cultured with and without tamoxifen and in the presence ofIL-3, IL-6 and SCF to terminate MYC-ER function for 7 days beforeanalyzing phenotypic markers. As shown in FIG. 4, a significantproportion of cells acquired B lineage markers including B220 (˜12%),CD19 (˜10%) and mIgM (˜10%). In addition, the inventors have been ableto generate the following lineages in vitro by withdrawal of 4OHT fromthe cultures: CD4+ ab T-cells, myeloid cells (Mac-1+), ter-119+erythroid progenitor cells, NK1.1 expressing cells, neutrophils (Gr-1+cells). Further experiments will assess the ability of these cells togive rise to other lineages, as well as the effect of altering thecytokine regimen on differentiation. Although the comparison has notbeen performed, the present inventors expect differentiation from younganimals, as compared to aged animals to be much more efficient in B cellproduction. To the best of the present inventors' knowledge, this is thefirst example of a conditionally immortal hematopoietic stem cell linethat can be induced to differentiate in vitro.

Example 2

The following example describes the results of adoptive transfer ofLT-HSC lines into lethally irradiated recipients.

If the HSC lines described in Example 1 are to be appropriate subjectsfor analysis of the basis of defective B cell lymphopoiesis in agedanimals, they should recapitulate the defect in vivo. The inventors havebegun to address this question by adoptive transfer of LT-HSC lines intolethally irradiated recipients. In initial experiments, lines from agedanimals (>60% ID⁻) were transferred along with RAG2^(−/−) bone marrow,and recipients were not treated with tamoxifen in order to silenceMYC-ER. Six weeks later recipient bone marrow and spleen cells wereharvested and the recovery and phenotype of GFP⁺ cells (GFP marks cellsderived from HSC lines) was analyzed (FIG. 5).

In the data from three mice presented in FIG. 5, one mouse received theaged HSC line ABM42, and two mice received aged HSC line ABM46.Depending upon the line transferred, 30 to 70% of cells in the lymphoidscatter gate were GFP⁺. As shown in FIG. 5, both lines tested (ABM46 andABM42) gave rise to B (CD19⁺) and T (TCR⁺, CD4⁺, CD8⁺) cells,macrophages (CD11b⁺) and granulocytes (GR1⁺). There was some recipientto recipient variation in the proportionality of these lineages.However, importantly, while both lines tested gave rise to mature CD4and CD8 single positive T cells (FIG. 7), B cell development did notproceed beyond the progenitor stage (FIG. 6). While B220⁺, CD19⁺ cellsdeveloped, they did not progress to the mIg⁺ stage. This is preciselythe outcome predicted by results of experiments involvingautoreconstitution and adoptive reconstitution using BM HSC fromimmunologically aged mice (Johnson, S. A., S. J. Rozzo, and J. C.Cambier, Aging-dependent exclusion of antigen-inexperienced cells fromthe peripheral B cell repertoire. J Immunol, 2002. 168(10): p. 5014-23).In other words, the same developmental arrest is observed when wholebone marrow from immunologically aged mice is used for transplantation.

The inventors have found that this system can be taken a step further,successfully re-establishing LT-HSC lines from bone marrow of adoptiverecipients of the original HSC lines (data not shown). This wasaccomplished simply by culturing bone marrow cells in stem cellcytokines plus tamoxifen to reactivate MYC. These cells are now growingand exhibit the original phenotype.

Example 3

The following example describes a method for reversibly immortalizingHSCs using a method conducted entirely in vitro.

In addition to the method for generating conditionally immortalized longterm HSC cell lines described previously herein, the inventors have beenable to carry out this procedure completely in vitro. The methoddescribed above relies upon introducing the transduced HSC's into mice,and inducing their transformation in vivo. The advantage of carryingthis procedure out in vitro is that every aspect of the process iscarried out in a controlled environment.

The method first includes the treatment of donor mice with5-fluorouracil (5-FU) in order to enrich for HSCs and induce these cellsto proliferate. 5FU enriched hematopoietic stem cells from the tibia andfemurs of mice were collected and then plated in 24 well tissue cultureplates in DMEM media containing 15% heat inactivated fetal calf serumand IL-3, IL-6 and SCF, at a density of 1.8-2.0×10⁶ cells per well. Thecells were subjected to three rounds of spin infection in order toretrovirally transduce the cells with retroviral vectors encoding MYC-ERand Bcl-2. Briefly, the cells were transfected with pMIG-MYC.ER orpMIT-Bcl2. The virus containing supernatants were collected andsupplemented with 4 μg/ml of polybrene and 10 mM HEPES, and passedthrough a 0.45 μm filter. The two different viral supernatants weremixed at a 1:1 ratio and added to the wells. The cells were thencentrifuged at 2000 rpm for one hour. The viral supernatants werereplaced at the end of each spin infection. 24 hours after the lastround infection, the levels of transduction were determined by flowcytometric analysis in order to determine the transduction efficiency.The transduced cells were then incubated in DMEM medium containing IL-3,IL-6, SCF and 10 nM 4OHT. The medium was replaced every 3 days andspecial emphasis was placed on ensuring a fresh supply of cytokines and4OHT. Cells are passed slowly, and as needed.

Using this in vitro approach, the inventors have been able to generateconditionally immortalized cell lines with the following combinations ofgenes: MYC-ER and Bcl-2; MYC-ER and hTERT (reverse transcriptasecomponent of the human telomerase); ICN-1-ER (ER-regulated activeelement of the intracellular portion of Notch-1) and Bcl-2; ICN-1-ER andhTERT; and MYC-ER and ICN-1-ER. The data presented in FIGS. 8-11 showthe initial characterization of most of these cell lines. They yieldedlines composed of c-kit+, Sca-1+, CD34−, flk2− cells, which is aphenotype that is consistent with the one presented by normal long-termhematopoietic stem cells. The data presented in FIGS. 8-11 is derivedfrom the flow cytometric analysis of retrovirally encoded reporter genes(GFP and thy1.1), as well as four markers for stem cells: c-kit, sca-1,CD34 and flk-2. The cell lines shown in FIGS. 8-11 had been in culturefor 5 weeks prior to phenotyping. These cells have been expanded anddivided in continuous culture for over 35 days to date.

Referring to FIG. 8, this figure shows the phenotypic comparison of celllines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with BCL-2 and MYC-ER and maintained incontinuous in vitro culture for >90 days. Shown is the phenotype ofrepresentative clones 3 (young) months after 90 days of continuous ofculture.

Referring to FIG. 9, this figure shows the phenotypic comparison of celllines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days. 5FU enrichedHSCs were retroviral transduced with pMIG-MYC and pMIT-Bcl-2 (toppanels), pMIG-MYC.ER and pMIG-hTERT (middle panels), or pMIG-ICN.1.ERand pMIT-Bcl-2. The cells were maintained in DMEM supplemented with 15%fetal calf serum, and a cocktail of IL-6, IL-3 and SCF. Shown is thephenotype of representative clones 3 (young) months after 90 days ofcontinuous of culture. The panels represent the results of the flowcytometric analysis for expression of the viral expression markers (GFPand Thy1.1), as well as four markers required to define long-term HSCsin mice, Sca-1, c-kit, CD34 and Flk-2. The four cell lines containedsubpopulations that retained the phenotypes of lt-HSCs (Sca-1+, c-kit+,CD34−, flk-2−).

Referring to FIG. 10, this figure shows the phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days. 5FU enrichedHSCs were retroviral transduced with pMIG-ICN.1.ER and pMIT-Bcl-2 (toppanels), pMIG-ICN.1 and pMIT-Bcl-2 (second row panels), or pMIG-ICN.1and pMIG-Bcl-2 (third row panels), or pMIG-hTERT and pMIT-Bcl-2 (bottompanels). The cells were maintained in DMEM supplemented with 15% fetalcalf serum, and a cocktail of IL-6, IL-3 and SCF. Shown is the phenotypeof representative clones 3 (young) months after 90 days of continuous ofculture. The panels represent the results of the flow cytometricanalysis for expression of the viral expression markers (GFP andThy1.1), as well as four markers required to define long-term HSCs inmice, Sca-1, c-kit, CD34 and Flk-2. The four cell lines containedsubpopulations that retained the phenotypes of lt-HSCs (Sca-1+, c-kit+,CD34−, flk-2−).

Referring to FIG. 11, this figure shows the phenotypic comparison ofcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days. 5FU enrichedHSCs were retroviral transduced with pMIG-MYC and pMIG-ICN.1 (toppanels), pMIG-MYC.ER and pMIG-ICN.1 (middle panels), or pMIG-ICN.1.ERand pMIG-MYC. The cells were maintained in DMEM supplemented with 15%fetal calf serum, and a cocktail of IL-6, IL-3 and SCF. Shown is thephenotype of representative clones 3 (young) months after 90 days ofcontinuous of culture. The panels represent the results of the flowcytometric analysis for expression of the viral expression markers (GFPand Thy1.1), as well as four markers required to define long-term HSCsin mice, Sca-1, c-kit, CD34 and Flk-2. The four cell lines containedsubpopulations that retained the phenotypes of lt-HSCs (Sca-1+, c-kit+,CD34−, flk-2−).

These cell lines have also been used to reconstitute cellularcompartments in vivo. Referring to FIG. 12, this figures shows theresults of in vivo reconstitution of T cell and B cell compartments fromcell lines derived from HSCs obtained from young C57/BL6 mice that wereretrovirally transduced with different combinations of oncogenes andmaintained in continuous in vitro culture for >90 days. Briefly, 5FUenriched HSCs were retroviral transduced with pMIG-ICN.1-ER andpMIG-hTERT (top panels), pMIG-MYC.ER and pMIG-hTERT (middle panels), orpMIG-MYC-ER and pMIT-Bcl-2 (lower panels). The cell lines weremaintained in DMEM supplemented with 15% fetal calf serum, and acocktail of IL-6, IL-3 and SCF. Lethally irradiated young C57/BL6 micewere reconstituted using bone marrow stem cells from Rag2−/− mice andLT-HSC lines generated in vitro. Six weeks later, bone marrow washarvested and stained with a panel of specific lineage markers. Thedevelopment of mature CD4 and B220 positive/GFP positive cells canreadily be observed. Data from four representative mice are presented inthis figure. In each group, approximately 30% of the mice retain GFPmarker.

Example 4

The following example describes an extension of the method forreversibly immortalizing human cord blood and bone marrow derived HSCsin vitro.

One additional application of this technology is the ability to expandhuman long-term hematopoietic stem cells in vitro through theirconditional immortalization. The inventors have therefore adapted the invitro method described in the previous examples for human cells with afew changes. First, the retroviruses are packaged preferably withamphotrophic envelopes in order to enable efficient transduction ofhuman cells. In addition, the source of the cells is human cord bloodobtained anonymously from the a cord blood bank, following all rules andregulations set forth by the Institutional Review Boards of theinventors' institutions. The resulting cells will express reporter genesthat may ultimately be useful for isolating a pure population by highspeed cell sorting. The inventors have noticed that many mature cellsresulting from the murine lt-HSC cell lines lose expression of thesurface markers, potentially due to the methylation of the retroviralgenome upon lineage determination and differentiation. The inventorsexpect to see similar behavior in the human cells, in which case thelt-HSCs and their prevalence in transplant recipients can be monitoredby the presence of reporter genes in such cells, in combination withcell surface markers for that population of cells.

Example 5

The following example describes an approach to the sequential excisionof the DNA fragments encoding MYC-ER and Bcl-2 from conditionallyimmortalized HSC cells.

In order to avoid taking the risk of introducing HSCs that harbortransgenes encoding MYC.ER and Bcl-2 into humans and/or mice, these twoDNA fragments will be excised using a bacterial recombinase approach.Two different recombinases will be used in order to allow control overwhich one of the two genes is excised at any one point in time. Twoexamples of such recombinases are the Cre and Flp recombinases. Briefly,the recognition substrate sequences (RSS's) for one of the recombinasesis introduced into the retroviral constructs such that they flank theopen reading frame of the oncogene, as well as the reporter gene (GFP orThy1.1). In this case, the cells are incubated in media containing aTat-Cre fusion protein. This recombinant protein has been previouslydescribed and shown to be able to passively enter cells, and mediateloxP site-dependent recombination of genomic DNA.

This approach will allow the achievement of a number of things in orderto enable the generation of many HSCs for differentiation in vitro andin vivo. First, the cells can gradually be weaned from the high levelsof proliferative and survival signals they had become accustomed toduring the conditional transformation process. Second, the cells can bere-adapted to depend on normal cytokines for their homeostatic functionsand differentiation. Third, the sequential loss of reporter expressionwill allow the definition of the status and degree of deletion of eachone of the genes in question. Accordingly, cells that express bothreporter genes (GFP and Thy1.1) harbor both sequences (MYC and Bcl-2,respectively), cells that express Thy1.1 but no GFP have successfullydeleted the MYC encoding sequences, but still contain Bcl-2 genes, andlastly, cells that do not express either GFP or Thy1.1 have deleted bothof those alleles. FIG. 13 represents this approach in a diagram.

In addition, this approach is tested in mice by obtaining 5FU enrichedBM-HSCs from a strain of mice in which the expression of a human MYCtransgene can be induced by the withdrawal of tetracycline and thepresence of a bacterial protein called tTA (tetracycline transactivatorprotein). The human MYC cDNA was cloned downstream of a tetracyclineregulatory transcription element (TRE). The TRE-MYC mice are treatedwith 5FU and used to harvest BM-HSCs. Those cells are transduced invitro with retroviruses expressing Bcl-2 and tTA (pMIT-Bcl2 andpMIG-tTA). The cells are cultured in the continuous presence ofDoxycycline in order to maintain the MYC transgene silent. Once thecells are analyzed by flow cytometry, they can be used fortransplantation back into mice that will not be maintained on adoxycycline containing diet (this is a more stable form of tetracyclineis normally used in vivo).

Once the lt-HSC cell lines are generated, the effect of culturing themin the presence of doxycycline in vitro will be examined in parallelwith MYC.ER harboring cell lines that will be cultured in the absence of4OHT. The protein levels of MYC are monitored by western blots andintracellular staining approaches throughout.

Example 6

The following example describes the generation of many hematopoieticlineages in vitro, following the withdrawal of 4OHT from the liquidtissue culture media.

The traditional methods used to determine the potency of an HSC involvethe use of semi-solid media (methycellulose) with defined cytokines inorder to potentiate the differentiation of HSCs into specific lineages.The inventors were interested in determining the pluripotency of thiscell population created using the method of the present invention invitro. In order to examine this issue, the ABM42 and ABM46 cell linesdescribed herein were maintained in media containing IL-3, IL-6 and SCF,but without 4OHT. In addition to the lineages that the inventors wereable to detect in the reconstituted mice (i.e., lymphoid, myeloid andgranulocytic), GFP+ cells could also be detected that expressed NK1.1 orter-119 (FIG. 14). The NK1.1 cells could either be NK-cell, or NK-Tcells. The ter-119 expressing cells are of the erythroid lineage. Thesefindings indicate that these cell lines are capable of giving rise toall of the elements of a normal hematopoietic system and that the cellswill be useful for generation of large quantities of specific elementsto be used for passive therapies. In addition, they will be of great useand importance to study the early events in hematopoiesis and toidentify novel therapy for therapeutic intervention in geneticdisorders, or complications that arise the normal course ofchemotherapy, or even infectious disease.

Example 7

The following example describes a method for high throughput screens ofsmall molecules or biological agents that induce or inhibitdifferentiation in conditionally transformed long term HSCs.

The following is a general method for screening small molecules orbiological agents that induce or inhibit HSC differentiation.Previously, these types of large screens were prohibited by the factthat large numbers of stem cells were unobtainable. With the presentinventors current ability to conditionally immortalize long term HSCs,it is now feasible to propose such technologies.

By way of example, one such method is a myeloid differentiation read-outthat has been adapted from Schneider, et al. (Schneider, T., andIssekutz, A. C. (1996). Quantitation of eosinophil and neutrophilinfiltration into rat lung by basic assays for eosinophil peroxidase andmyeloperoxidase. Application in a Brown Norway rat model of allergicpulmonary inflammation. J Immunol Methods 198, 1-14). Briefly,conditionally transformed long term HSCs are plated in 96 well, flatbottom plates at various concentrations of cell numbers (usually2×10⁴-5×10⁴ cells/well). The screens are carried out either in completemedia (DMEM +15% heat inactivated fetal calf serum, 1×penicillin/streptomycin, 1×1-glutamine and 1× non-essential amino acids,supplemented with IL-3, IL-6 and SCF) with added 4OHT in order tomaintain the cells in an undifferentiated state, or in the absence ofadded 4OHT in order to induce differentiation. These conditions havebeen shown to give rise to Mac-1+ cells, consistent with a myeloiddifferentiation pattern. Additional cytokines can be added to directdifferentiation in specific paths, although this system can also be usedto screen for specific functions of a panel of cytokines. In thisinstance, the complete media will be added without supplementation withIL-3, IL-6 and SCF, but instead with the given cytokines to be tested orused to direct differentiation (e.g., CSF-1, G-CSF, GM-CSF, EPO, TEPO,etc.).

Small molecules, biological agents or positive control substances (e.g.,Arsenic 0₃) are titrated across the 96 well plate and incubated with theltHSCs for time frames ranging from 24 to 72 hours, or longer, if neededand as determined based on the agents or molecules to be tested. Afterincubation, the cells are washed with PBS and resuspended in PBS forovernight storage at −80° C. to lyse the cells. The cells are thenthawed at room temperature and the plates are centrifuged for 10 min at3,000 rpm. The supernatant is then transferred to a new 96 well plateand mixed with tetramethybenzidine (TMB) for 40 min. The reaction isstopped with 4NH₂SO₄ and the O.D. is read at 450 nm. This type ofhigh-throughput assay can be used to test small molecules or biologicalagents for the ability to induce or block the differentiation ofconditionally transformed long term HSCs into a wide variety of celltypes. Results of these screens can then further be tested for theability to induce or inhibit HSC differentiation in vivo. Variations onthis assay format will be apparent to those of skill in the art and areencompassed by the present invention.

Example 8

The following example describes the use of the method of the inventionto generate cell lines of an intermediate hematopoietic lineage.

The following protocol can be used to induce the development of celllines representing intermediate stages of hematopoietic lineagedevelopment following transplantation of conditionally immortalizedlt-HSC cell lines into lethally irradiated mice. First, 10³-10⁵conditionally transformed lt-HSC cell lines generated according to themethod of the invention are transferred into cohorts of lethallyirradiated recipient mice. The transplants will also include 10⁵Rag-1^(−/−) cells as carriers in order to ensure the initial survival ofthe irradiated mice. The mice are treated with weekly injections of 1 mgtamoxifen, intraperitoneally, in order to immortalize partiallydifferentiated cells derived from the conditionally transformed lt-HSCcell lines. Injections begin either 3 days-1 week after the initialtransplant, or 8 weeks after the transplant, once the mice have beenfully reconstituted by the conditionally transformed lt-HSC cell lines.Cells are collected from the spleen and bone marrow cells from micethree days after treatment with tamoxifen, or when they show clinicalsigns associated with leukemias. The cells are cultured in either thestandard bone marrow culture conditions with 4-OHT (DMEM, 15% fetal calfserum, pen/strep, L-glut, non essential amino acids, IL-3, IL-6 andSCF), or in the presence of other cytokines and medium used fordifferent hematopoietic cell types. Cell lines are frozen and/orexpanded, and cell lines are also single-cell cloned by limitingdilution and defined by PCR amplification of proviral integrations,frozen, and then characterized for surface marker expression by flowcytometry. These types of approaches are used for both murine and humanctlt-HSC cell lines, using either NOD/SCID mice as the recipients, orneonatal Rag-1−/− mice, which will be given intrahepatic injections.

Example 9

The following example describes the use of the method of the inventionand the adoption of protocols used to generate mature CD4+ αβ T-cells invitro to develop cell lines representing intermediate stages of T-celldevelopment.

In this experiment, conditionally immortalized lt-HSC cell linesgenerated according to the method of the invention are plated in thepresence of the normal cytokine cocktail, supplemented with IL-7 andwithout tamoxifen. Parallel cultures are established on a layer of OP-9stromal cells that express Jagged, a Notch-1 ligand. Cells are stainedfor T-cell lineage markers every 48 hours after the cultures areinitiated to monitor for signs of T-cell development. The wells thatshow signs of T-lineage commitment and development are switched to mediacontaining tamoxifen in order to stabilize the phenotype and establishcell lines. The resulting cell lines are expanded, cloned andcharacterized as described in Example 8. The T-cell lines arespecifically stained for individual TCR-Vβ alleles in order to determinetheir T-cell receptor repertoire usage. Some mature T-cell lines, orcell lines representing progenitor populations, are transplanted intoRag-1^(−/−) mice in order to evaluate their ability to conform to normaltolerance and homeostatic mechanisms in vivo, as well as their abilityto further differentiate in vivo, when appropriate. Finally, theirability to respond to antigenic stimulation is evaluated in vitro and invivo.

Example 10

The following example describes the use of the method of the inventionand the adoption of protocols used for the directed differentiation ofHSCs into myeloid cell lineages to develop intermediate developmentalcell lines and myeloid leukemia models.

In this experiment, conditionally immortalized lt-HSC cell linesgenerated according to the methods of the present invention are platedin the presence of the normal cytokine cocktail, supplemented with G-CSFand without tamoxifen. Cells are stained for myeloid lineage markersevery 48 hours after the cultures are initiated to monitor for signs ofmyeloid development. The wells that show signs of myeloid lineagecommitment and development are switched to media containing tamoxifen inorder to stabilize the phenotype and establish cell lines. The resultingcell lines are expanded, cloned and characterized as described inExample 8. Some of the resulting cell lines are transplanted back intomice in order to monitor their ability to repopulate Op/Op mice (mutantmice that naturally lack macrophages). Those cell lines are alsotransplanted into wild type mice that will be maintained on tamoxifenthroughout, in order to determine if these cell lines will also giverise to myeloid leukemias similar to human AML, CML and APL. These noveltumors provide novel models for preclinical therapeutics.

Example 11

The following example describes the generation of Human adult ctlt-HSCcell lines and examination of their pluripotential in vivo usingNOD/SCID or RAG^(−/−) xenotransplant models.

In this experiment, CD34+ cells (from mobilized blood or cord blood) aretransduced in vitro with retroviral vectors encoding MYC-ER, Bcl-2 andGFP (for later detection of transplanted cells), packaged usingamphotrophic envelopes (according to the methods of the presentinvention). lt-HSC are selected by propagation in vitro in the presenceof 4OHT and growth factors, as described above using murine HSCs.Pluripotency of the selected cells is evaluated by transplantation oflt-HSC lines into sublethally irradiated NOD/SCID or NOD/SCID/β-2M^(−/−)or Rag-1^(−/−) or Rag-2^(−/−) mice, followed 6-12 weeks later byanalysis of all blood cell lineages by immunofluorescence flowcytometry. More particularly, following the generation of ctlt-HSC celllines using the method of the present invention, one can use twodifferent and complimentary approaches to examine their pluripotency. Ina first approach, varying amounts of clonal ctlt-HSC cell lines areintroduced into sublethally irradiated NOD/SCID mice orNOD/SCID/β-2M^(−/−) mice. In this instance, 10³-10⁵ cells derived from ahuman ctlt-HSC cell lines are transferred intravenously after the miceare subjected to a sublethal irradiation regimen (0.3 Gy). The mice areanalyzed for reconstitution at 6-12 weeks after transplantation. Second,10³-10⁵ cells derived from a human ctlt-HSC cell lines are introducedinto the liver of neonatal Rag-1^(−/−) or Rag-2^(−/−) mice by directinjection. Those xenotransplants will also be analyzed for appropriatereconstitution 6-12 weeks after transplantation.

Example 12

The following example describes the use of conditional approaches toabrogate expression of MYC and Bcl-2 from the ctlt-HSCs aftertransplantation.

In this experiment, viruses (viral vectors) used to transform stem cellsare re-engineered to contain two loxP sites flanking the MYC-ER, Bcl-2and GFP open reading frames (ORFS). When the cells are transplanted, aregulated form of Cre or CRE-TAT fusion protein will be used to deletethe oncogene-encoding sequences, thus eliminating risk of insert-drivenmalignancy in recipients. This approach is first developed in mice, thenapplied to human lt-HSCs.

In a second approach, lt-HSCs from TRE-MYC mice are used to generate thecell lines with retroviruses that encode Bcl-2 or rtTA. These aretransplanted into mice. Mice are fed Doxycycline to abrogate theexpression of MYC. One can use lt-HSCs obtained from TRE-MYCxTRE-Bcl-2bigenic mice that can be transduced with a pMIG-rtTA retrovirus toeliminate MYC and Bcl2 expression.

Example 13

The following example describes the use of HIV-1 Tat protein fusionswith MYC and/or Bcl-2 to attain conditional transformation withoutgenetic modification of the lt-HSCs.

MYC-Tat and Bcl-2-Tat fusion proteins are generated and purified usingestablished protocols. The fusion proteins are tested by treatment ofcells in which one can easily assay the effects of overexpressed Bcl-2(e.g., activated T cells, B-cell lymphoma cell lines that are renderedresistant to BCMA-Fc, etc.) or MYC (e.g., anergic B-cells, naïveT-cells, activated T-cells). Combinations of MYC-Tat and Bcl-2-Tatproteins are used to allow propagation of lt-HSCs prior totransplantation. This approach is readily developed and tested in themouse system, then applied to human.

The entire disclosure of each of U.S. Provisional Patent ApplicationNos. 60/728,131 and 60/765,993 is incorporated herein by reference.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. A method of producing a population ofconditionally immortalized hematopoietic stem cells, comprising: a)introducing into a plurality of hematopoietic stem cells: i. anexogenously synthesized first polypeptide comprising: a MYC polypeptidethat has MYC activity; and ii. an exogenously synthesized secondpolypeptide comprising: a Bcl-2 polypeptide; so as to produce at leastone conditionally immortalized hematopoietic stem cell; b) culturing theplurality of hematopoietic stem cells in media comprising at least oneof: IL-3, IL-6, stem cell factor, thrombopoietin, Flt3 ligand, or acombination thereof such that a population of conditionally immortalizedhematopoietic stem cells is produced, and wherein the first polypeptide,the second polypeptide, or both the first and second polypeptidescomprise a TAT sequence.
 2. The method of claim 1, further comprising(c) removing the first polypeptide such that the population of the stemcells differentiate.
 3. The method of claim 1, further comprising (c)removing the first polypeptide such that the population of the stemcells differentiate and, culturing the population of stem cells in thepresence of at least one cytokine that directs differentiation towards aspecific cell type.
 4. The method of claim 1, wherein the Mycpolypeptide is a c-MYC polypeptide, a n-MYC polypeptide, a s-MYCpolypeptide, or a 1-MYC polypeptide.
 5. The method of claim 1, whereinthe first polypeptide further comprises the hormone-binding domain ofthe human estrogen receptor.
 6. The method of claim 5, furthercomprising expanding the population of conditionally immortalized stemcells by contacting the plurality of stem cells with: estradiol (E2),4-hydroxytamoxifen (4-0HT), or both.
 7. The method of claim 1, whereinthe plurality of stem cells are isolated from umbilical cord blood, bonemarrow, or a combination thereof.
 8. The method of claim 1, furthercomprising modifying one or more genes of the plurality of stem cells.9. A method of producing a population of conditionally immortalizedhematopoietic stem cells, comprising: a) introducing into a plurality ofhematopoietic stem cells: i. an exogenously synthesized firstpolypeptide comprising: a MYC polypeptide that has MYC activity; and ii.an exogenously synthesized second polypeptide comprising: a Bcl-2polypeptide; so as to produce at least one conditionally immortalizedhematopoietic stem cell; b) culturing the plurality of hematopoieticstem cells in media such that a population of conditionally immortalizedhematopoietic stem cells is produced, and wherein the first polypeptide,the second polypeptide, or both the first and second polypeptidescomprise a TAT sequence.